优秀论文类别：临床血液学检验882576血小板淋巴细胞比和中性粒细胞淋巴细胞比在前列腺癌和良性前列腺增生患者中辅

助筛查前列腺癌患者的作用研究1

魏高辉郑州大学第一附属医院检验科，河南省检验医学重点实验室，郑州 450052

[摘要]目的评估 PLR和NLR在 PCa和 BPH初诊患者中筛查 PCa患者的价值。方法收集我院 2012 年至 2015 年 PCa 和 BPH 住院患者 96 人；分析两组患者初诊PLR， NLR 差异性，评估其诊断价值。结果 PCa 患者的 PLR 中位数为103.6 （ IQR,88.1-120.7 ） ， BPH 患 者 的 PLR 中 位 数 为 127.2 （ IQR,91.0-183.8），PCa初诊患者的 PLR明显低于 BPH初诊患者（P=0.011）；PCa患者NLR中位数 1.95（IQR,1.4-2.8），BPH患者 NLR中位数 2.6（IQR,1.7-4.5），PCa初诊患者 NLR明显低于 BPH初诊患者（P=0.008）；应用 ROC曲线分析，PLR的曲 线 下 面 积 为 0.664 （ 95%CI ， 0.554-0.773 ） ， NLR 的 曲 线 下 面 积 为0.643（95%CI，0.536-0.750），两指标均具有诊断价值；ROC曲线计算 PLR和NLR的最佳 cutoff值，PLR小于 145，NLR小于 2.55作为在 PCa和 BPH初诊患者中筛查 PCa患者的依据，PLR小于 145作为诊断标准的敏感度高达 92.9%。结论PLR，NLR可作为有价值指标在 PCa和 BPH初诊患者中筛查 PCa患者。[关键词]PLR；NLR；前列腺癌；良性前列腺增生Platelet lymphocyteratioand neutrophillymphocyteratioin patients withprostatecancer and benignprostatic hyperplasiascreeningfor prostate cancer[Abstract] Objective:For prostate cancer ( PCa) and benign prostatic hyperplasia (BPH) newly diagnosed patients, assess platelet lymphocyte ratio (platelet-lymphocyte ratio, PLR) and neutrophil lymphocyte ratio (neutrophil to lymphocyte ratio, NLR) screening value ofPCa patients.Methods:The database contains 96 PCa andBPH patients; analyzed two groups of patients with newly diagnosed PLR, NLR and evaluated the diagnostic value.Results:Themedian PLR of PCa patients was103.6 (IQR, 88.1-120.7) and BPH patients was 127.2 (IQR, 91.0-183.8) (P = 0.011); Themedian NLR ofPCa patients was 1.95 ( IQR, 1.4-2.8) and BPH patientswas 2.6 (IQR, 1.7-4.5) (P = 0.008); the area under the curve(AUC) recorded for PLR was0.664 (95%CI, 0.554-0.773) and for NLR was 0.643 (95%CI, 0.536-0.750); calculation of the ideal cutoff valuesof PLR and NLR, PLR is less than 145 andNLR is less than 2.55 as the basis for screening PCa patients in PCa and BPH patients. Conclusion:PLR, NLR can be used as a valuable indicator for PCa patients in PCa and BPH patients.[Keywords] PLR；NLR；prostate cancer；benign prostatic hyperplasia前列腺癌（ prostatic cancer， PCa）和良性前列腺增生（ benign prostatic

hyperplasia，BPH）严重影响老年男性的健康及生活质量。前列腺疾病住院患者多1

为 PCa和 BPH。两者均为慢性前列腺疾病，经历早期病变，缓慢进展逐渐演变而来[1]；二者的初始临床表现相似，都包括排尿障碍，尿路梗阻等。PCa多发于前列腺背侧及外侧部分的外周带，潜伏期长，进展缓慢；BPH多发于前列腺尿道周围移行带，为多发结节增生组织，并逐渐增大以致引起临床症状[2]。前列腺活检病理检查是 PCa和 BPH诊断的金标准，因其具有侵入性，使应用

受到极大的限制；为了避免不必要的活检，其他检查如直肠指检，前列腺特异性抗原（prostate specific antigen,PSA），经直肠超声检查都用来综合诊断及判断是否需要活检[3]。PSA作为一种器官特异性的肿瘤标志物，现在广泛应用于 PCa的筛查，然而在 BPH中该指标同样会升高。循证医学证明 PSA筛查带来的获益并不明确，甚至可能导致过度医疗，因此建议 PSA不再作为 PCa筛查常规项目[4]。

PCa和 BPH都与机体的慢性炎症关系密切。Nickel JC等分析发现 PCa和 BPH患者前列腺病理标本都不同程度的存在慢性炎症反应 [5]。且炎症指标 C反应蛋白水平增高[6]，血小板增多[7]，淋巴细胞减少[8]被作为肿瘤病人预后差的依据。血小板淋巴细胞比（ platelet-lymphocyte ratio， PLR）和中性粒细胞淋巴细胞比（neutrophil to lymphocyte ratio，NLR）作为一种简单易获取的炎症生物学指标被用于肿瘤患者的预后判断，高水平的 PLR，NLR表明肿瘤患者预后不良 [9, 10]。PLR可用于鉴别子宫内膜癌和子宫内膜增生症[11]。由此我们思考 PLR，NLR是否可用于 PCa与 BPH的鉴别。本研究为回顾性研究，针对初诊患者评估 PLR，NLR在 PCa和 BPH初诊患者

中筛查 PCa患者的价值。1材料与方法1.1研究对象 选取郑州大学第一附属医院 2012年至 2015年的因前列腺疾病住院患者，根据患者住院期间的病例信息，获取前列腺活检，开放性前列腺手术，经尿道前列腺电切术的前列腺组织病理结果，分为 PCa和 BPH患者。本研究纳入标准：初诊患者并获取初次来院时血常规检查结果（未经治疗）。初诊患者定义：因前列腺疾病初次来我院就诊，未经治疗且在我院明确诊断的患者。选择初诊患者是为了排除治疗因素对 PLR和NLR结果的影响。排除有急性炎症，感染，糖尿病，高血压，高血脂，急慢性肾衰竭，慢性肝病，炎症性肠病，结缔组织病等疾病的患者。共计有 96名患者纳入本研究，其中 PCa初诊患者 43人，BPH初诊患者 53人。1.2 数据收集入院时患者有多次血常规结果的取距离就诊时间最近的结果。血常规指标：白细胞计数，淋巴细胞计数，血小板计数，血小板平均体积（mean platelet volume，MPV），血小板分布宽度,结果来自我院检验科（贝克曼五分类血液分析仪）。PCa患者的全身核素骨显像检查结果，PCa患者病理学 Gleason评分结果；PCa和 BPH患者年龄，PSA数据；PSA数据包括总 PSA（本研究 PSA即指总PSA），游离 PSA，总 PSA与游离 PSA比值。1.3 统计分析连续数值变量的数据以中位数和四分位间距（interquartile range,IQR）表示。对连续数值变量的统计学分析采用非参数统计的双侧Mann-Whitney U检验；

根据受试者工作曲线（Receiver operating characteristic,ROC），用 ROC曲线敏感度和特异度之和的最大值，确定 cutoff值，即 PLR<145和 NLR<2.55作为在 PCa和BPH初诊患者中诊断 PCa的标准。其它指标如年龄，游离 PSA，总 PSA与游离PSA比值，白细胞计数，淋巴细胞计数，血小板计数，MPV，血小板分布宽度的统计学分析采用双侧Mann-Whitney U检验。使用 SPSS17.0软件进行统计学分析，P<0.05视为差异有统计学意义。2结果2.1 病人一般特点病人一般特点见表 1。PCa患者确诊年龄中位数 70岁（IQR:65-74）；PCa患

者的 Gleason评分 38人，其中评分小于 7的 11人（28.9%），评分大于等于 7的27人（71.1%）。BPH患者确诊年龄中位数 70岁（IQR：61-77）。2.2 PLR，NLR在初诊前列腺癌和良性前列腺增生患者中的差异性比较及在初诊前列腺癌患者转移与否、Gleason评分差异性比较

对初诊前列腺疾病患者 96人（PCa患者 43人，BPH患者 53人）的初诊PLR,NLR进行统计学分析，结果见表 2，PCa患者 PLR中位数 103.6(IQR,88.1-120.7)，BPH患者 PLR中位数 127.2(IQR,91.0-183.8)，PLR在 PCa和 BPH初诊患者间差异有统计学意义（P=0.011），PCa初诊患者的 PLR明显低于 BPH初诊患者；PCa 患者 NLR 中位数 1.95(IQR,1.4-2.8)，BPH 患者中 NLR 中位数 2.6(IQR,1.7-4.5)，NLR在 PCa和 BPH初诊患者间差异有统计学意义（P=0.008），PCa初诊患者 NLR明显低于 BPH初诊患者。上述结果表明 PLR，NLR可区分 PCa和 BPH初诊患者。

根据 PCa患者全身核素骨显像检查结果，对 PLR，NLR在有无骨转移 PCa患者间的差异性进行分析，结果 PLR（P=0.836）、NLR（P=0.773）在有无骨转移初诊 PCa患者中差异性无统计学意义；PCa患者 Gleason评分小于 7和大于等于 7的 PLR（0.814）、NLR（0.366）差异性无统计学意义（表 2）。由此可见PLR，NLR不能有效区分 PCa患者的肿瘤侵袭性及进展状况。2.3 PSA,PLR,NLR在前列腺癌和良性前列腺增生初诊患者中诊断前列腺癌患者的ROC曲线及曲线下面积分析

对 PSA，PLR，NLR区分 PCa和 BPH的价值进行方法学评估，根据 ROC曲线评价 PSA,PLR,NLR诊断 PCa和 BPH患者中的 PCa患者的价值，其中 PSA的曲线下面积（ area under the curve， AUC）为 0.765（ 95%置信区间 [confidence interval，CI]（图 1A），0.659-0.870）；PLR 的 AUC 为 0.664（95%CI，0.554-0.773） (图 1B)；NLR 的 AUC 为 0.643（ 95%CI， 0.536-0.750）（图 1C）；PLR，NLR可作为有价值指标在 PCa和 BPH初诊患者中筛查 PCa患者。ROC曲线计算 PLR的 cut off值为 145，NLR的 cut off值为 2.55，以此分析 PLR，NLR在PCa和 BPH初诊患者中筛查 PCa患者的诊断价值。

2.4 PLR，NLR在前列腺癌和良性前列腺增生初诊患者中筛查前列腺癌患者的诊断价值评估

PLR<145在 PCa和 BPH初诊患者中诊断 PCa的阳性预测值，阴性预测值，敏感度和特异度分别为 56.5%，88.0%，92.9%和 42.3%；NLR<2.55在 PCa和 BPH初诊患者中诊断 PCa 的阳性预测值，阴性预测值，敏感度和特异度分别为55.6%，70.7%，71.4%和 54.7%（表 3）；由此结果可知 PLR作为一个独立的指标诊断疾病敏感度高达 92.9%，说明该指标的漏诊率低，可作为疾病的筛查指标。相比之下NLR作为诊断指标的价值比 PLR低。2.5 非参数检验统计分析前列腺癌和良性前列腺增生患者其它相关参数的差异性其它相关指标在 PCa和 BPH初诊患者中差异性的比较见表 4；其中年龄，白

细胞计数，血小板计数，血小板分布宽度在 PCa和 BPH初诊患者间无统计学差异；淋巴细胞计数，游离 PSA，总 PSA与游离 PSA比值，MPV在 PCa和 BPH初诊患者间差异有统计学意义，提示这些指标具有潜在的诊断价值。3 讨论PCa和 BPH的发生都与炎症密切相关。研究表明感染及不良饮食与 PCa的发生密切相关[12]，上述因素可导致慢性炎症，损伤细胞 DNA，导致增生性炎症萎缩，增加患癌风险；Steiner GE等对 BPH患者前列腺病理切片进行分析发现，绝大多数患者前列腺中存在炎性浸润细胞，尤其是 CD4+辅助性 T细胞大量增加[13]，这些炎性细胞直接刺激基质和上皮细胞增生，增加炎性细胞因子释放，使前列腺体积增大。目前研究的热点是炎症指标对肿瘤患者预后的评估。研究表明炎症指标 C反应蛋白升高，血小板增多，中性粒细胞增多及淋巴细胞减少表明肿瘤患者的不良预后[14-

18]。PLR和 NLR是一种简单非侵入性易获取的生物学标志物，作为炎症指标广泛用于癌症患者的预后判断，如卵巢癌 [19]，食管癌 [20]，鼻咽癌 [21]及前列腺癌 [22]等。PLR和NLR也可以作为 PCa风险评估的依据，McDonald AC等对前列腺无症状人群 PSA进行分析发现，同那些正常的 PSA人群相比，高水平的 PSA人群存在高水平 C反应蛋白，血浆纤维蛋白原，PLR，NLR，预示患癌风险增高[23]。PLR可用于对侵入性检查的可行性评估，Smith RA等利用 PLR联合 CA199来判断对壶腹部肿瘤患者进行腹腔镜检查的必要性，结果表明 PLR结合 CA199可显著降低不必要的腹腔镜检查数量[24]。本研究结果，PLR和NLR在 PCa患者中均显著低于 BPH患者。结果表明 PCa患者炎症水平不高，这可能与肿瘤免疫相关，PCa的发展经历：正常——炎症性萎缩后增生（Proliferative inflammatory atrophy,PIA）——低度前列腺上皮内瘤变 (Low-grade PIN, prostatic intraepithelial neoplasia)——高度前列腺上皮内瘤变 (High-grade PIN, prostatic intraepithelial neoplasia)——PCa(前列腺癌)。在这个过程中免疫系统未有效清除瘤变细胞及之后的癌细胞，免疫系统不能发挥正常的免疫功能。BPH患者存在更强的炎症反应，这一点在 Robert G的研究中可有体现，其对 282名BPH患者前列腺病理切片免疫组化进行分析发现，绝大部分的炎性浸润细胞表达 T

细胞，B细胞及巨噬细胞表面标记，IPSS评分与前列腺体积成正相关，前列腺增生组织存在大量炎性细胞浸润[25]。 本研究对 PLR和 NLR在 PCa和初诊患者中筛查 PCa患者的能力进行了方法学评价，ROC曲线计算 PLR和 NLR的 cutoff值，PLR为 145，NLR为 2.55；PLR<145在 PCa和 BPH中诊断 PCa的敏感度高达 92.9%。由此可见，PLR和 NLR可用于PCa筛查。Mehmet Kaynar[26]等的研究发现 PLR在 PCa和 BPH的差异性，在 PSA大于 10ng/ml时，PLR在 PCa中显著低于 BPH患者，得出可用 PLR区分 PCa和BPH，NLR在两者之间无差异性，其研究对象并非前列腺初诊患者，不能排除药物及手术对结果的影响，且未对 PLR区分 PCa和 BPH的价值进行方法学评价。基于我国 PCa和 BPH患者就诊晚，病情重，PSA水平普遍高的事实，本研究以初诊患者作为研究对象，发现 PLR，NLR在 PCa和 BPH初诊患者之间均存在差异，这与Mehmet Kaynar的研究结果不同；对 PLR，NLR进行方法学评价，计算出 PLR的 cutoff值为 145，NLR的 cut off值为 2.55，同时对两指标的诊断价值进行评价，对 PLR，NLR筛查 PCa的价值有了清楚的认识。因此本研究是首次对 PLR，NLR在 PCa和 BPH患者中筛查 PCa患者进行方法学及诊断价值评估。 对 PCa 和 BPH 其它相关指标进行分析发现 MPV 在两者之间存在差异性（P=0.013），Jong-Han Lee等研究发现MPV，NLR在脑梗塞患者中于 CRP显著相关，可结合 CRP对脑梗塞患者进行病情评估[27]。MPV在 PCa和 BPH中的差异性是否存在更深层次的联系，将是我们下一步研究的重点。本研究同样存在局限性，首先为了研究 PCa和 BPH初诊患者，剔除了大量手术患者，激素去势治疗患者，及放疗患者，样本量偏少；其次，认定初诊患者是根据病人病例，不能排除一些患者就医前服药或其他治疗因素对本研究的影响；最后因本院慢性前列腺炎病人很少，未将此病纳入本研究。因此要明确 PLR，NLR在 PCa和 BPH初诊患者中的诊断价值，还需要更大样本量及更严格病人筛选标准的大规模随机对照临床试验。综上所述，本研究发现 PLR小于 145，NLR小于 2.55可作为 PCa和 BPH初诊患者中筛查 PCa患者的标准，并发现 MPV在 PCa和 BPH初诊患者中的差异性，为今后在 PCa和 BPH患者中筛查 PCa患者提供参考。表 1 病人基本信息

PCa初诊患者特点初诊病人 43

确诊年龄，中位数（四分位间距） 70（65-74）确诊时 PSA水平<10 ng/ml 6（14.3%）10–20 ng/ml 10（23.8%）

>20 ng/ml 26（61.9%）Gleason 评分<7 11（28.9%）>7 27（71.1%）BPH初诊患者特点初诊患者 53

确诊年龄，中位数（四分位间距） 70（61-77）确诊时 PSA水平<10 ng/ml 26（65.0%）10–20 ng/ml 9（22.5%）>20 ng/ml 5（12.5%）表 2.PLR，NLR在初诊前列腺癌和良性前列腺增生患者中的差异性比较及在初诊前列腺癌患者转移与否、Gleason评分差异性比较

初诊 PLR 初诊 NLR

n PLR中位数(IQR) P PLR中位数(IQR) P

PCa 43 103.6(88.1-120.7) 0.011 1.95(1.4-2.8) 0.008

BPH 53 127.2(91.0-183.8) 2.6(1.7-4.5)

PCa

无转移 33 104.8(87.1-119.5) 0.836 2.0(1.4-2.6) 0.773

骨转移 10 102.2(91.9-127.9) 1.8(1.6-3.1)

Gleason评分<7 9 108.9(77.2-112.2) 0.814 1.7(1.3-2.5) 0.366

>7 34 99.7(89.5-123.9) 2.0(1.6-3.0)

前列腺癌（PCa）;良性前列腺增生（BPH）四分位间距（IQR）; P值采用双侧Mann-Whitney U 检验。

表 3 PLR，NLR在前列腺癌和良性前列腺增生初诊患者中筛查前列腺癌患者的诊断价值评估

PCa BPH 诊断价值(%)

初诊 PLR 阳性预测值(56.5%)

<145(n=69) 39 30 阴性预测值(88.0%)

敏感度(92.9%)

>145(n=25) 3 22 特异度(42.3%)

初诊NLR 阳性预测值(55.6%)

<2.55(n=54) 30 24 阴性预测值(70.7%)

敏感度(71.4%)

>2.55(n=41) 12 29 特异度(54.7%)

前列腺癌（PCa）;良性前列腺增生（BPH）

表 4 非参数检验统计分析前列腺癌和良性前列腺增生患者其他相关参数的差异PCa* BPH* P 值

1白细胞计数 6 .3(5.0-7.3) 6.4(5.0-7.9) 0.524

1血小板计数 182(145-218) 186(158-205) 0.802

1淋巴细胞计数 1.7(1.4-2.1) 1.4(1.1-1.9) 0.014

确诊年龄 70(65-74) 70(61-77) 0.949

fPSA(ng/ml) 3.4(1.2-12.1) 1.0(0.3-2.0) <0.001

F/P 0.15(0.10-0.23) 0.21(0.15-0.29) 0.032

MPV(fL) 8.1(7.8-8.4) 8.8(7.8-8.9) 0.013

PDW(%) 16.3(15.9-16.9) 16.5(16.1-16.8) 0.294*中位数(四分位间距)；1(×109/L)；fPSA,游离 PSA；F/P，fPSA/tPSA7；血小板平均体积，MPV；血小板分布宽度，PDW。P值采用双侧Mann-Whitney U 检验。

图 1. PSA,PLR,NLR在前列腺癌和良性前列腺增生初诊患者中诊断前列腺癌患者的ROC曲线及曲线下面积

曲线下面积（AUC）,置信区间（CI）参考文献[1] Sciarra A, Di SF, Salciccia S, Autran GAM, Gentilucci A, Gentile V.

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类别：临床血液学检验842444骨髓细胞形态学检查联合ALP、PSA在前列腺癌骨转移导致全血细胞减少诊断中

的意义焦扬　刘冰　吕晓娴　盛家和　许青霞＃

郑州大学附属肿瘤医院检验科 河南郑州 450008摘要 　 目的：探讨骨髓细胞形态学检查联合 ALP、PSA在前列腺癌骨转移导致全血细胞减少诊断中的意义。方法：回顾分析 2006年 1月至 2014年 1月我院 84例出现全血细胞减少的 PCa患者，按是否骨转移分为,前列腺癌(PCa)骨转移组（53例）和非骨转移组（31例），PCa骨转移与非骨转移组间均值比较采用 t检验，计数资料采用卡方检验，均以 P<0.05为差异有统计学意义。结果：PCa骨转移与非骨转移组血清 ALP 和 PSA 含量比较，差异显著，有统计学意义 (P<0.05)；以ALP≧120U/L和 PSA≧50ng/ml分别为判断前列腺癌骨转移标准分组，两组分别进行比较，差异显著，有统计学意义(P<0.05)；三种方法联检的方法与它们单独对PCa 骨转移的诊断的比较，灵敏度和阴性预测值显著提高（P<0.05），特异度显著减低（P<0.05），阳性预测值降低不明显；结论：三种方法联检虽然显著提高了PCa 骨转移的临床诊断的灵敏度，但其误诊率也明显提高，由于癌细胞多呈灶性增生，为提高临床 PCa 骨转移的临床诊断的准确性，一次骨穿涂片阴性不应除外转移癌，应多次多部位穿刺，在放射学检查或核素骨扫描异常处穿刺进行骨髓细胞形态学检查以提高 PCa 骨转移诊断准确性。关键词：骨髓细胞形态；前列腺癌；骨转移；碱性磷酸酶；前列腺特异性抗原The clinical significance of bone marrow cell morphology combined ALP and PSA in pancytopenia caused by bone metastasis of prostate cancerJiao Yang, Liu Bing, Lv XiaoXian, Sheng Jiahe, Xu Qingxia＃ Clinical Laboratory , The Tumor Hospital of Zhengzhou University，Zhengzhou , Henan 450008 , ChinaAbstract Objective: To investigate the clinical significance of bone marrow cell

morphology combined ALP and PSA in pancytopenia caused by bone metastasis of

prostate cancer. Methods: A total of 84 prostate cancer patients with pancytopenia in our

hospital from Jan. 2006 to Jan. 2014 was analysed, according to whether they had

the bone metastasis, the patients were divided into prostate PCa bone metastasis

group (53 cases) and non-bone metastasis group (31 cases), the mean values of PCa

bone metastasis group and non-bone metastasis group were compared with t test, and

count data by chi-square test, the difference was statistically significant with P < 0.05.

Results: Comparing the content of serum ALP and PSA in PCa bone metastasis

group and non-bone metastasis group, the difference was significant, with statistical

significance(P<0.05); according to the standard of ALP≧120U/L and PSA≧50ng/ml, the

patients were divided into PCa bone metastasis group and non-bone metastasis group,

two groups are compared, the difference was significant, with statistical

significance (P<0.05); comparing the united three methods with the three methods alone

for the diagnosis of PCa bone metastasis, the sensitivity and negative predictive

value increased significantly (P<0.05), specificity was significantly

decreased (P<0.05), the positive predictive value was not reduced significantly.

Conclusion: Although the united three methods significantly increased the sensitivity

of the clinical diagnosis of PCa metastasis to bone, but its misdiagnosis rate was

increased significantly, too. Because cancer cells showed focal hyperplasia, in order to

improve the accuracy of clinical diagnosis for bone metastasis of PCa, a bone

puncture smear negative should not except for metastatic carcinoma,

multiple sites should be punctured, in order to improve the diagnostic accuracy of

PCa for bone metastasis, bone puncture sites shoud be where had abnormal radiological

examination or radionuclide bone scanning.

Key words：Morphology of bone marrow cells; Prostate cancer; Bone metastasis;

Alkaline phosphatase; Prostate specific antigen

　　骨髓细胞形态检查具有操作简单、快捷，细胞形态、结构清晰等优点，是血液病最基本且常用的检查方法，但骨髓穿刺涂片易受操作、穿刺部位、麻醉不当、“干抽”、“稀释”和制片水平的影响，以及转移癌细胞往往呈灶性分布，病变区域骨髓纤维化发生率较高，导致骨髓穿刺涂片不能反映骨髓的真实情况，造成临床的漏诊或误诊。恶性肿瘤发生骨髓转移，浸润并破坏骨髓造血组织，导致血液学发生改变。前列腺癌(prostatecancer,PCa)在我国男性泌尿生殖系统恶性肿瘤中发病率较高，仅次于膀胱癌及肾癌，且 PCa临床早期无症状,发现时已多为进展期，有24%～35%的 PCa患者初诊时就已转移,而且约占 70%为骨转移[1,2]。本文回顾分析84例 PCa患者骨髓细胞形态学检查联合 PSA和ALP检测对 PCa骨转移导致全血细胞减少的诊断意义。资料和方法一、对象 　

　　回顾分析 2006年 1月至 2014年 1月我院 84例出现全血细胞减少的 PCa患者，均经穿刺活检或手术后病理诊断为 PCa，且均有完 善的骨髓细胞学检查和PSA、ALP检测结果，并已排除由于其它原因导致ALP升高患者，年龄 51～92岁,平均 74岁。通过 ECT、X 线、CT、PET－CT或者骨髓活检等方法确定 PCa患者是否骨转移并分组,PCa骨转移组 53例，而非骨转移组 31例。二、标本采集与处理

1.髂前或髂后上棘穿刺抽取骨髓液涂片 6～8 张，干燥后采用瑞氏染色法染骨髓片 2 张，40分钟后，用水冲净玻片，凉干后备用。

2.清晨空腹促凝管肘正中静脉采血 2mL，采用 3000转/分离心分离血清备用；三、方法 　　1.先用光学显微镜低倍镜观察整张涂片，然后再用油镜进一步检查确认，发现成堆分布的非造血细胞即为转移癌细胞，即诊断为 PCa骨转移。　　2.采用罗氏 Cobas501全自动生化分析仪检测患者血清 ALP含量，参考文献，PCa患者 ALP＞120U/L即可诊断为 PCa骨转移[3]；采用罗氏 E170全自动电化学分析仪检测 PSA含量，参考文献，PCa患者 PSA＞50ng/ml即可诊断为 PCa骨转移[4]。四、统计学处理　　应用 SPSS 15.0统计学软件进行处理，计量资料以 ±s表示，骨转移与非骨转移组间均值比较采用 t检验，计数资料采用 χ2检验。均以 P<0.05为差异有统计学意义。结果

1.PCa细胞骨髓转移，癌细胞常成团出现于片尾、片头或两边，总的特点都不同于骨髓内的正常造血细胞；多数癌细胞成簇出现，少者 4—5个成群，多者几十个细胞成簇，彼此间界限不清，形态大小不一，但也有单个散在分界清晰的癌细胞，直径一般大于骨髓细胞，但巨核细胞除外；细胞呈多形性变，可见空泡、脂肪变性，核质比例变大，核的形状与结构异常，核大，核染色质增粗，可见核分裂象，双核、多核易见，核仁巨大且不规则，胞质浓深蓝色，有的有空泡和颗粒；细胞体积大，呈现癌细胞特有的细胞体积大，核大，核仁大而明显，核染色质增粗深染，排列极性紊乱，大小不一等特点（具体如图 1，图 2）。本研究通过骨髓形态学镜检发现，PCa骨转移组 53例患者，有 37例发生骨转移，非骨转移组 31例患者，未发现骨转移患者，敏感度为 69.8%，灵敏度为 100%，与文献[5]报道相符。

　图 1　PCa骨转移低倍镜 10×10 倍　　　　图 2　PCa骨转移油镜 10×100 倍2.PCa骨转移组和非转移组ALP和 PSA含量比较，转移组 ALP和 PSA含量显

著高于非转移组(P<0.05)，如表 1 表 1 PCa骨转移组和非转移组ALP和 PSA含量比较( ±s) Group Case ALP(U/L) PSA(ng/ml)Metastasis 53 168±190 809±1631Non-metastasis 31 80±36 64±156PCa非转移组与骨转移组比较 P<0.05

3.不同血清 PSA水平 PCa骨转移情况对比，不小于 50ng/ml组与小于 50ng/ml组相比较，有显著性差异(P<0.05)，如表 2表 2 不同血清 PSA水平 PCa骨转移情况对比(n%)

PSA(ng/ml) Case Metastasis Non-metastasis≧50 51 45 6<50 33 8 25小于 50ng/ml组与不小于 50ng/ml组相比较 P<0.05

4.不同血清 ALP水平 PCa骨转移情况对比，不小于 120U/L组与小于 120U/L组相比较，有显著性差异(P<0.05)，如表 3表 3 不同血清ALP水平 PCa骨转移情况对比(n%)

ALP(U/L) Case Metastasis Non-metastasis≧120 36 29 7<120 48 24 24小于 120U/L组与不小于 120U/L组相比较 P<0.05

5.骨髓细胞形态学检查、ALP、PSA与三者并联应用对 PCa骨转移比较，三项联检灵敏度和阴性测值显著提高(P<0.05)，而特异度显著降低(P<0.05)，阳性预测值降低不显著。 表 4 骨髓细胞形态学检查、ALP、PSA与三者并联应用对 PCa骨转移比较(%)Target Case Sensitivity Specificity PPV NPVMorphology 37 69.8 100 100 66.0ALP 36 54.7 77.4 80.6 50.0

PSA 51 84.9 80.6 88.2 75.5United three 52 98.1 51.6 77.6 94.1PPV(Positive predictive value),NPV(Negative predictive value)三者并联应用与形态学检查、ALP和 PSA比较 P<0.05

讨论骨髓具有丰富的血流，这使得多种肿瘤均可转移至骨髓，有的甚至是肿瘤细胞

唯一的转移场所。前列腺癌骨转移是常见的骨髓转移癌之一 [6]。前列腺癌原发灶较为隐蔽，体积小，症状不明显，有时临床上虽未发现明显体征，但血象已经发生了某些改变。由于血运丰富，前列腺癌细胞转移至骨髓后，癌细胞会大量增殖，抑制正常的造血系统，癌细胞骨髓侵犯会引起骨髓抑制性、破坏性因子的早期释放，而正常造血刺激因子的释放受抑制，引发患者全血细胞减少。血液学改变有时是前列腺癌骨转移早期主要的临床表现，根据文献报道，多数人认为前列腺癌骨转移引起全血细胞减少是由于癌细胞浸润增殖，排挤骨髓血细胞，破坏骨髓血液屏障，从而引起造血细胞被的损伤，进一步机体代偿性出现髓外造血和骨髓纤维化所致。骨髓涂片如找到非造血细胞的癌细胞，不管是成堆分布还是单个散在，均是骨

髓转移癌的肯定性诊断依据。虽然骨髓细胞形态检查具有操作简单、快捷，细胞形态、结构清晰等优点，是血液病最基本且常用的检查方法，但是骨髓穿刺涂片易受操作、穿刺部位、麻醉不当、“干抽”、“稀释”和制片水平的影响，以及转移癌细胞往往呈灶性分布，病变区域骨髓纤维化发生率较高，导致骨髓穿刺涂片不能反映骨髓的真实情况，造成临床的漏诊或误诊，本研究人工骨髓涂片镜检敏感度为69.8%，灵敏度为 100%，与文献[5]报道相符。血清 PSA是早期发现 PCa敏感性很高的肿瘤标志物之一，目前临床上将其结

合直肠指检普查早期 PCa作为诊断 PCa的有效的一线方法，近年来，又将其用于PCa骨转移的辅助诊断指标[7]。作为 PCa骨转移诊断与鉴别诊断的一项重要指标，PSA有较高的特异性和敏感性，本研究以 PSA≧50ng/ml为判断前列腺癌骨转移的节点，骨转移组与非转移组相比有显著差异（P<0.05），诊断前列腺癌骨转移的灵敏度为 84.9%，特异度为 80.6%，对可疑前列腺癌的患者定期行 PSA 跟踪检查，有助于早期发现前列腺癌骨转移，与文献相符[8]。

ALP是一组磷酸单酯水解酶,在碱性条件下,能催化磷酸基团水解和转移。它几乎存在于机体各个组织中,但以骨骼和肝脏含量最高[9]。血清 ALP 主要由成骨细胞分泌,当某些疾病(如骨折、恶性肿瘤骨转移等)造成骨骼损伤时,成骨细胞分泌 ALP 增加以促进磷酸盐沉积,加速骨骼修复,可以引起血清 ALP 活性升高[10]，所以血清ALP 活性升高是骨骼病变的重要诊断指标。本研究结果显示 ,以ALP≧120U/L为判断前列腺癌骨转移的节点，PCa骨转移组血清 ALP活性平均水平明显高于非骨转移组（P<0.05）,诊断前列腺骨髓转移的灵敏度为 54.7%,特异度为 77.4%,与文献报

道一致[11]，可见血清 ALP 活性测定可以作为判断骨转移的参考指标，但敏感度较低。骨髓细胞形态，血清 PSA和血清ALP 各有其优点和不足，血清 PSA同时有较

高的灵敏度和特异度，骨髓细胞形态有很高的特异度，但灵敏度较低，而血清ALP灵敏度较低，而特异度也不是很高，为提高 PCa 骨转移诊断的正确性，本文采用这三种方法的并联联检的方式来帮助诊断 PCa 骨转移，结果显示联检的方法与它们单独对 PCa 骨转移的诊断的比较，灵敏度和阴性预测值显著提高（P<0.05），但特异度显著减低（P<0.05），阳性预测值降低不明显。 综上所述，骨髓细胞形态，血清 PSA和血清 ALP联检虽然显著提高了 PCa 骨

转移的临床诊断的灵敏度，但其误诊率也明显提高，分析其原因可能两种：一是并联联检造成的灵敏度提高而特异度降低，二是血清 PSA和血清 ALP诊断 PCa 骨转移的节点选取不够准确，需进一步分析来得到更加准确的血清 PSA和血清 ALP诊断 PCa 骨转移的节点。在此基础上，由于癌细胞多呈灶性增生，临床上一次骨穿涂片阴性不能除外转移癌，应多次多部位穿刺，应在放射学检查或核素骨扫描异常处穿刺进行骨髓细胞形态学检查以提高 PCa 骨转移诊断准确性。四　参考文献[1] 杨桂凤,左书耀,王国明，等.骨显像诊断前列腺癌骨转移与病理分级及 PSA的关系探讨 [J].中国临床医学影像杂志,2010,21(1):72-73.[2] Saylor PJ,Keating NL,Smith MR.Prostate cancer survivorship:prevention and treatment of the adverse effects of androgen deprivation therapy[J].J Gen Intern Med,2009,24(Suppl 2):S389-S394.[3] 邱梅婷.PSA和 ALP联合检测在早期前列腺癌骨转移诊断中的应用[J].河北医科大学 　学报,2012,33(6):728-730.[4] 邝永龙,王德林,吴小候.ALP,PSA及其相关指标与前列腺癌骨转移的关系[J].现代泌尿生殖肿瘤杂志,2010,2(5):208-212.[5] 张宗,雷敏,赵佳,等.恶性肿瘤骨髓转移的诊断和细胞形态学分析[J].淋巴瘤·白血病,2011,20(12):753-755.[6] 赵华山,常见恶性肿瘤临床指引[M].武汉:湖北科技出版社,2006:262.[7] Lamb DS,Slamney D,Smart R,et a1.Prostate cancer the new evidence base for diagnosis and treat mean[J].Pathology,2007,39(6):537．[8] Strope SA,Andriole GL.Prostate cancer screening:current status and future perspectives[J].Nat Rev Urol,2010,7(9):487-493.[9] Leeming DJ,Koizumi M,Byrjalsen I,et al.The relative use of eight collagenous and noncollagenous markers for diagnosis of skeletal metastases in breast,prostate,or lung cancer patients[J].Cancer Epidemiol Biomarkers Prev ,2006,15(1):32-38.[10] 陈旭芳.血清碱性磷酸酶检测恶性肿瘤骨转移诊断价值的探讨[J].中华肿瘤防治杂志,2009,16(12):953-954.[11] 范晓莉.恶性肿瘤骨转移诊断与治疗进展[J].临床合理用药,2009,2(12):92-94.

类别：临床基础检验872245Comparative proteomic analysis of Trichinella spiralis muscle larvae and intestinal infective larvae彭若玉郑州大学第一附属医院，河南郑州，450052Abstract

Trichinella spiralis (T. spiralis) is one of the most widespread parasites which can be found in many species of carnivores and omnivores. Trichinella infection is initiated by the consumption of undercooked meat containing T. spiralis larvae. Herein, isobaric tagging reagents for relative and absolute quantification (iTRAQ)-based liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis was used to identify the effect of ABZSO on the proteome of T. spiralis muscle larvae in vitro. A total of 3,795 proteins were quantified from 22,974 unique peptides. Comparative proteomics analysis showed that 417 proteins were markedly differentially expressed in ABZSO-treated larvae, of which 213 were up-regulated and 204 were down-regulated. Gene ontology annotation and KEGG pathway analysis showed that most of the differentially expressed proteins were involved in cell apoptosis, signal pathway, amino acid metabolism, protein synthesis/assembly/degradation and other biological processes.

IntroductionTrichinella spiralis (T. spiralis) is one of the most widespread parasites which can

be found in many species of carnivores and omnivores. Trichinella infection is initiated by the consumption of undercooked meat containing T. spiralis larvae. Once ingested, the muscle larvae (ML) are liberated from their capsules by the action of host’s digestive enzymes and activated to intestinal infective larvae (IIL) by intestinal content or bile. Then, the IIL invade and migrate through intestinal epithelium cells (IECs) to continue their life cycle. Therefore, the invasion of T.spiralis IIL is the first and crucial step in the process of Trichinella infection.

Because there is no stylet or dentate structure in the mouth of T. spiralis larvae, the invasion of T. spiralis IIL is not mechanical damage, but more likely out of the molecular mechanism. From previous studies, it is known that there are lots of genes and proteins were differentially expressed  in T. spiralis ML and IIL, and some T. spiralis proteins were also identified from host intestinal epithelial cells after cultured with T. spiralis larvae. We can speculate that the changed T.spiralis proteins play an important role in the invasion, development and interactions of host and T. spiralis. However, up to now, the complete and detailed comparative proteomic analysis of T. spiralis ML and IIL has not been reported.In the present study, the proteomic change of T. spiralis ML and IIL was investigated by isobaric tagging reagents for relative and absolute quantification (iTRAQ) -based liquid chromatography and tandem mass spectrometry (LC/MS-MS) analysis. This study will be helpful in providing important hints for uncovering the pathogenic mechanism of T. spiralis larvae and finding new targets for Trichinella prevention and treatment.Materials and MethodsParasites, experiment animals and cell lines

The isolates (ISS534) of T. spiralis used in this study were obtained from a domestic pig in Nanyang City of Henan Province, China. Specific pathogen-free BALB/c mice (six-week-old, female) were purchased from the Experimental Animal Center of Henan Province (Zhengzhou, China). Human colonic epithelial cell line HCT-8 was obtained from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. All animal procedures reported herein were approved by the Life Science Ethics Committee of Zhengzhou University (Permission No. SYXK 2007-0009).Collection of ML and IILT. spiralis ML were obtained from experimentally infected mice by artificial digestion and collected by Baermann's method after three settlings, and the process was repeated once more. Then the larvae were washed throughly in sterile phosphate-buffered saline (PBS). Half of the larvae were stored at −80℃ as the control group (D1，D2), and the other part were cultured at 37 with fresh bile for 2 h and then co-incubated℃  with HCT-8 cells for 6 h. Then, the IIL larvae were collected as the experimental group (S1，S2).Protein preparation and SDS-PAGE analysisSDT buffer (4% SDS, 150 mM Tris-HCl, 100 mM DTT, pH 8.0) in presence of protease inhibitor (Thermo, USA) was added into the frozen parasite samples, and then the lysate was homogenized by MP homogenizer (24×2, 6.0M/S, 60s, twice). The larval fragments were sonicated (99 times 3-s cycle, 100 W, 0 ), and boiled for 15min. After centrifuged℃ at 14,000g for 40 min, the supernatant was collected and filtered with 0.22 µm filters. All samples were quantified using the BCA Protein Assay Kit (Bio-Rad, USA), then stored at -80 .℃20 µg of proteins for each sample were mixed with 5× loading buffer respectively and boiled for 5 min. The proteins were separated on 12.5% SDS-PAGE gel (constant current 14 mA, 90 min). Protein bands were visualized by Coomassie Blue R-250 staining.Protein preparation and iTRAQ labelingLarval protein digestion was performed using the filter-aided sample preparation method. 200 μg of proteins for each sample were incorporated into 30 μl SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl pH 8.0). The detergent, DTT and other low-molecular-weight components were removed using UA buffer (8 M Urea, 150 mM Tris-HCl pH 8.0) by repeated ultrafiltration (Microcon units, 10 kD). Then 100 μl iodoacetamide (100 mM IAA in UA buffer) was added to block reduced cysteine residues and the samples were incubated for 30 min in darkness. The filters were washed with 100 μl UA buffer three times and then 100 μl Dissolution buffer (0.5M triethylammonium bicarbonate, 0.2% SDS) twice. Finally, the protein suspensions were digested with 4 μg trypsin (Promega, USA) in 40 μl DS buffer overnight at 37 °C, and the resulting peptides were collected as a filtrate. The peptides of each sample were desalted on C18 Cartridges (Sigma, USA), concentrated by vacuum centrifugation and reconstituted in 40 µl of 0.1% (v/v) formic acid. According to the manufacturer’s instructions (AB SCIEX, USA), The resulting peptide samples D1, D2, S1, and S2 were labeled with iTRAQ reagents 114, 115, 116, and 117, respectively, and then combined into one sample mixture and dried for strong cation exchange (SCX) fractionation. Strong Cation Exchange (SCX) ChromatographyiTRAQ labeled peptides were fractionated by SCX chromatography using the AKTA Purifier system (GE Healthcare, USA). The dried peptide mixture was reconstituted and

acidified with buffer A (10 mM KH2PO4 in 25% of ACN, pH 3.0) and loaded onto a PolySULFOETHYL 4.6 x 100 mm column (5 µm, 200 Å, PolyLC Inc, Maryland, USA). The peptides were eluted at a flow rate of 1 ml/min with a gradient of 0%–8% buffer B (500 mM KCl, 10 mM KH2PO4 in 25% of ACN, pH 3.0) for 22 min, 8–52% buffer B during 22-47 min, 52%–100% buffer B during 47-50 min, 100% buffer B during 50-58 min, and buffer B was reset to 0% after 58min. The elution was monitored by absorbance at 214 nm, and fractions were collected every 1 min. The collected fractions were desalted on C18 Cartridges and concentrated by vacuum centrifugation.HPLC and LS-MS/MS analysisEach fraction was injected for nanoLC-MS/MS analysis. The peptide mixture was loaded onto a reverse phase trap column（Thermo Scientific Acclaim PepMap100, 100μm*2cm, nanoViper C18 ） connected to the C18-reversed phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3μm resin) in buffer A (0.1% Formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% Formic acid) at a flow rate of 300 nl/min controlled by IntelliFlow technology. The linear gradient was 0-35% buffer B for 50 min, 35-100% buffer B for 5 min, hold in 100% buffer B for 5 min.LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific) that was coupled to Easy nLC (Thermo Fisher Scientific) for 60 min. The mass spectrometer was operated in positive ion mode. MS data was acquired using a data-dependent top10 method dynamically choosing the most abundant precursor ions from the survey scan (300–1800 m/z) for HCD fragmentation. Automatic gain control (AGC) target was set to 3e6, and maximum inject time to 10 ms. Dynamic exclusion duration was 40.0 s. Survey scans were acquired at a resolution of 70,000 at m/z 200 and resolution for HCD spectra was set to 17,500 at m/z 200, and isolation width was 2 m/z. Normalized collision energy was 30 eV and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%. The instrument was run with peptide recognition mode enabled.Protein identification and quantificationMS data were acquired at data-dependent acquisition conditions, and the detailed information about the related methods has been described previously. MS/MS spectra were searched using MASCOT engine (Matrix Science, UK) embedded into Proteome Discoverer 1.4 (Thermo, USA) against Uniprot Trichinella database (33 825 sequences, download at April 15, 2016) and the decoy database. The following parameters were set for protein identification ： enzyme = trypsin, max missed cleavages = 2, fixed modifications: carbamidomethyl (C), iTRAQ4plex (N-term), iTRAQ4plex (K), variable modifications: oxidation (M), iTRAQ4plex (Y), peptide mass tolerance = ± 20 ppm, fragment mass tolerance = 0.1 Da, FDR≤0.01. The protein ratios were calculated as the median of only unique peptides of the protein, and all peptide ratios were normalized by the median protein ratio, which should be 1 after the normalization.Statistical and bioinformatic analysesStatistically significant changes were identified with fold change and p value. A protein, which has a fold change of >1.20 or <0.83 and with a p value<0.05, was considered significantly differentially expressed. The AmiGO tool of gene ontology platform (http://www.geneontology.org/) was applied to extract the functional protein analysis. The Search pathway tool of Kegg Mapper platform

(http://www.genome.jp/kegg/mapper.html) was used to extract pathway analysis. The enrichment analysis was performed by Fisher’s exact test.Quantitative real-time PCR Transcription of ten genes in the larvae was evaluated by quantitative real-time PCR, and the primers of them were designed using the Primer6.0 software and listed in Table 1. G3PDH was used as the internal control (Ren et al., 2013). Total RNA of the T. spiralis larvae under identical conditions of larvae used in the iTRAQ proteomic analysis was isolated using TRIzol reagent (Invitrogen, USA). Total cDNA was produced using PrimeScript™RT reagent Kit with gDNA Eraser (Takara, Japan) according to the manufacturer’s protocols. The qPCR reaction mixture contained 2 μL diluted cDNA, 10 μL of 2× SYBR Premix Ex Taq (Takara, Japan), 0.8 μL of forward primer, 0.8 μL of reverse primer, 0.4 μL of 50×Rox Reference Dye II, and 6 μL of nuclease-free water to produce the final volume of 20 μL. The qPCR reactions were performed on a 7500 Fast Real-Time PCR System (Thermo, USA). Relative expression levels of the ten genes, normalized to G3PDH, were calculated using the comparative Ct (2-ΔΔCt) method. Every sample was repeated in triplicate, and each experiment was repeated three times.ResultsSDS-PAGE analysis20 µg of proteins for each sample were separated on 12.5% resolving gels, and distinct band patterns for T. spiralis ML and IIL were clearly revealed by SDS-PAGE analysis. The protein samples were abundant and suitable for subsequent trypsin digestion and LC–MS/MS analysis. Proteomic analysis of differentially expressed proteins of T.spiralis ML and IILSoluble proteins were obtained from T. spiralis ML and IIL, and compared by iTRAQ-based LC-MS/MS analysis. In total, 3790 proteins with quantitative information were quantified based on 25928 peptides. Among these proteins, 442 proteins of T. spiralis were significant differentially expressed in IIL, with 215 proteins up-regulated and 227 proteins down-regulated. Theoretical MW and pI distribution of the identified proteins of T.spiralis ML and IIL The theoretical molecular weight (MW) and isoelectric point (pI) of the identified proteins were calculated using the compute pI/MW tool (http://cn.expasy.org/tools/pi_tool.html) according to protein amino acid sequence. Distribution of MW and pI of the T. spiralis proteins identified was analyzed. MW ranged from 3.39 kDa to 1778.75 kDa, 43 (67.2%) proteins were distributing in a range of 10–70 kDa. The protein pIwas between 4.47 and 10.92, with 26 proteins (40.6%) distributed within the range of pI 5–6. Furthermore, approximately 3 (2.48%) proteins with higher pI (more than 10), which could not be separated by 2-DE, were also identified by LC–MS/MS. Gene ontology analysis for the differentially expressed proteins of T.spiralis ML and IILGene Ontology (GO) signatures of 54 out of the 64 proteins identified were available. To further understand the functions of the proteins identified in this study, we queried against the InterPro databases and those resultant proteins were classified into cellular component, molecular function and biological process according to GO hierarchy using WEGO.

The cellular components ontology refers to the place in the cell where a gene product is active. T. spiralis proteins in HCT-8 cells cultured with the infective larvae were annotated as being associated with cellular components. Of these proteins, 46 occur in the cell (GO:0005623) and 35 occur in the organelle (GO:0043226), with the remainder occurring in the macromolecular complex (GO:0032991), membrane-enclosed lumen (GO:0031974) and synapse (GO:0045202) which indicate the active communications among the neurons in the infective larvae.For the molecular function ontology, seven subcategories were assigned, of which the activities of binding (GO:0005488, 43 proteins, 79.6% of 54 annotated peptides) and catalytic activity (GO:0003824, 23, 42.6%) were the two major molecular function categories. Most of the assigned binding activity could be assigned to RNA binding (GO:0003723), protein binding(GO:0005515), nucleotide binding (GO:0000166) and, to a lesser extent, transcription factor binding (GO:0008134). The proteins in the catalytic activity group also can be classified into subgroups based on the specific functions. The majority of proteins are related to hydrolase activity and transferase activities. The groups with much fewer terms included oxidoreductase activity (GO:0016491), activation of MAPK activity (GO:0000187) and helicase activity (GO:0004386). In the biological process category, a large proportion of 54 proteins of T. spiralis were related to cellular process (GO:0009987), metabolism (GO:0008152), and regulation of biological process (GO:0050789), response to stimulus (GO:0050896) and signaling (GO:0023052). Less commonly, the proteins were involved in immune system process (GO:0002376), developmental process (GO:00332502), death (GO:0016265) and reproduction (GO:0000003). Most of the cellular and metabolic processes were related to synthesis and degradation of macromolecules, particularly carbohydrates, nucleotides and proteins, which might be associated with the invasion and development of T. spiralis infective larvae.

KEGG pathway analysis for the differentially expressed proteins of T.spiralis ML and IILKEGG can be used for systematic analysis of gene functions, which is linked with genomic information combined with information stored in the PATHWAY database. The KEGG pathway is an aggregate of pathway maps showing the present knowledge on the molecular interaction networks. In the present study, to find out the possible signaling pathways underlying the ABZSO treatments on T. spiralis muscle larvae, the protein sequences were aligned in KEGG GENES database using KAAS (KEGG Automatic Annotation Server), and then annotated to biochemical pathways using the KO number of similar proteins. Totally, the results showed that the identified 119 proteins were involved in 172 pathways, respectively. The top twenty-one represented KEGG maps were listed in Fig. 6, and the top eight ones were lysosome (9 members), pathways in cancer (8 members), mRNA surveillance pathway (7 members), apoptosis (7 members), pathogenic escherichia coli infection (6 members), RNA transport (6 members), endocytosis (6 members), and pyrimidine metabolism (6 members). All the detailed informations of differentially expressed proteins involving in the KEGG pathways were shown in Table S3.

Fig. 7 was the pathway map of apoptosis, which covered several related proteins, including tubulin alpha-3 chain (A0A0V1C0B7, A0A0V1C0Z2, A0A0V1BAN8), cathepsin F (E5SFB3), Cathepsin B-like cysteine proteinase 3 (A0A0V1C148), apoptosis regulator protein (E5S8V8), and putative C2 domain protein (E5SIH8). Besides, a variety of pathways associated with apoptosis were also reflected in the diagram, such as natural killer cell mediated cytotoxicity, TNF signaling pathway, protein processing in endoplasmic reticulum, calcium signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, NF-κB signaling pathway, and p53 signaling pathway. What′s more, the pathway of lysosome was showed in Fig. 8, which covered proteins including deoxyribonuclease II family protein (E5S4S7), niemann-Pick C1 protein (A0A0V1ARI2), proactivator polypeptide (A0A0V1B3G8), cathepsin F (E5SFB3), Cathepsin B-like cysteine proteinase 3 (A0A0V1C148), lysosomal-associated transmembrane protein 4A (E5SLQ5), histidine acid phosphatase family protein (E5SJV5, E5S3R2), putative uncharacterized protein (Q962V2), and the related pathways of lysosome were endocytosis, regulation of autophagy, snare interactions in vesicular transport, glycosaminoglycan degradation and other glycan degradation. In addition to KEGG, a protein-protein interaction network was constructed using IntAct Database (http://www.ebi.ac.uk/intact/main.xhtml), which was shown in Fig. S1.

类别：临床微生物检验878106Characterization of teicoplanin non-susceptible Staphylococcus epidermidis clinical

isolates belonging predominantly to ST267

C. Liu· L. Ming* Department of clinical laboratory, The First Affiliated Hospital of Zhengzhou University, Henan province Key Laboratory of Medicine, Zhengzhou, People's Republic of China

Abstract Objective Staphylococcus epidermidis is the main cause of bacteremia and

infections of indwelling, glycopeptide is often considered the choice of empirical drug for

the treatment of staphylococcal infections. Methods In the present study, 12 teicoplanin

non-susceptible Staphylococcus epidermidis clinical isolates were collected during Jan

2013 to Oct 2013. Results All strains carried the mecA gene, and showed heterogeneous

resistance to vancomycin using the PAP/AUC method. Multilocus sequence type (MLST)

revealed eight isolates belonged to the same ST type, ST267. Pulsed field electrophoresis

(PFGE) with SmaI endonuclease showed four distinct pulsotypes. Conclusion Our study

demonstrated that ST267 was the most epdemic clone among teicoplanin non-susceptible

S. epidermidis and identified a potential endemic clone in this region, which is believed

to be the first report that the ST267 clone has spread in China. Our findings revaled that

strengthen monitoring of drug resistance of Staphylococcus epidermidis to glycopeptides

is urgent needed, and heightened measures should be taken to control the further spread

of ST267 clone.

Keywords Staphylococcus epidermidis; teicoplanin resistance; ST267; MLST

IntroductionStaphylococcus epidermidis (S. epidermidis) is the most frequently encountered CoNS

(coagulase-negative staphylococci) species on human skin and the premier cause of

CoNS infections[1-4]. In recent years, S. epidermidis infection is a growing concern, due

to the high prevalence rate of meticillin-resistant Staphylococcus epidermidis and their

persistence on indwelling devices, often leading to the device replacement, which causes

longer hospitalization, increased rates of morbidity, and higher cost of treatment[4-6]. To

date, Glycopeptide antibiotics have often been used in empirical and rational therapy for

serious infections caused by coagulase-negative staphylococci because of the high

incidence of methicillin resistance CoNS clinical isolates. Since first reported in Japan in

1997[7], heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and

vancomycin-intermediate Staphylococcus aureus (S. aureus) have been detected

throughout the world, which have been recognised as a diagnostic and therapeutic

challenge. Teicoplanin-resistant CoNS was first reported in the UK and the USA in

1986[8], two years later, the appearance of glycopeptide resistant enterococci was

documented [9]. However, major interest regarding the basis of acquisition of resistance

to glycopeptides remained centered on enterococci and S. aureus, and data regarding

glycopeptide resistant in coagulase negative staphylococcus remain rare.

The possibility of acquired glycopeptide resistance among enterococci is often

considered to be exogenous origin (such as the VanA and VanB types), in contrast,

glycopeptide resistance among staphylococci appears to be endogenous. Despite the

mechanism of glycopeptide resistance in CoNS is still unclear, ultrastructural and

biochemical analysis observed a significant thickened cell wall and the production of a

large number of peptidoglycan in previous studies[10-12].

At present, only sporadic reports of teicoplanin non-susceptible CoNS strains, the

multilocus sequence typing results demonstated that the most represented sequence type

was ST2 and ST59 in China[13]. In the current report, we observed 12 teicoplanin non-

susceptible CoNS strains collected between Jan 2013 to Oct 2013 at the First Affiliated

Hospital of Zhengzhou University, which is a 7000-bed university teaching hospital.

Interestingly, our study found that ST267 was the most prevalent clone, which is believed

to be the first report that ST267 clone has spread in China. In addition, antibiotic

resistance profiles, systematically molecular characterization and mechanisms of

glycopeptide resistance were further evaluated.

Materials and methods

Bacterial isolates and detection of antibiotic susceptibility A total of 12 non-duplicate teicoplanin non-susceptible Staphylococcus epidermidis

clinical isolates were collected from a large tertiary teaching hospital (The First Affiliated

Hospital of Zhengzhou University, Zhengzhou, China) with 7000 beds located in north-

central China from Jan 2013 to Oct 2013. All Staphylococcus epidermidis isolates were

identified using the VITEK 2 compact (bioMerieux, France) and 16S rRNA gene

sequencing. MICs (Minimum inhibitory concentrations) of antibiotics were detected

using the broth microdilution method according to the CLSI (Clinical Laboratory

Standards Institute) guidelines (2014). The reference strain ATCC 29213 was used as the

control. E-test analysis of teicoplanin was performed using E-test strips (AB bioMérieux,

France) according to the manufacturer's requirements. Tigecycline result was interpreted

as recommended by the European Committee on Antimicrobial Susceptibility Testing

(EUCAST 2013).

Modified population analysis profiling/area under the curve (PAP/AUC)Modified PAP/AUC was performed as described by Wootton et al[14] with a few

modifications. Briefly, Following 24 hours of incubation in TSB (Tryptone Soya Broth)

(Oxoid, Basingstoke, England), Aliquots (100μ L) of cell suspension with turbidity

equivalent to 0.5 McFarland standard and of serial 10-fold dilutions were inoculated onto

Brain Heart Infusion agar (Oxoid, Basingstoke, England) plates containing 2 mg/L, 4

mg/L, 6 mg/L, 8mg/L, 10 mg/L,12mg/L, 14 mg/L and 16 mg/L vancomycin. Colonies

were counted after 48 hours. Control strains [ATCC 29213 (methicillin-susceptible S.

aureus, MSSA), Mu3 (heterogeneous vancomycin- intermediate S. aureus, hVISA) and

Mu50 (vancomycin-intermediate S. aureus, VISA)] were used for comparison in each

run.

DNA isolationAll isolates were cultured on Muller-Hinton agar and incubated overnight at 37 .℃

Genomic DNA was extractd using the Puregene Yeast/Bact. Kit (Qiagen, Valencia, CA,

USA) according to the manufacturer's instruction for Gram-positive bacteria. DNA

samples were stored at -20 refrigerator until test.℃

Molecular typing

Multiplex PCR was used to amplify the mecA gene and determine the staphylococcal

chromosome cassette mec (SCCmec) type(I-IV) of all S. epidermidis isolates, according

to the method published by Milheirico et al[15]. Multilocus sequence typing (MLST) was

examined as previously described[16]. Nucleotide sequences were analyzed via the

MLST website (http://www. mlst. net).

Detection of the Van genesThe vancomycin-resistant genes (VanA, VanB, VanC1, VanC2 and VanC3) were

determined using multiplex PCR as previously described [17]. The PCR was performed

as follows: initial denaturation at 94 for 5 min , then 35 cycles at 94 for 1 min,℃ ℃

58 for 1 min, and 72 for 1 min, followed by a final 10 min extension at 72 .℃ ℃ ℃

Autolysis analysisTriton X-100 stimulated autolysis was measured as described previously[18]. When

bacterial suspensions were grown to an OD600 of ~0.5, the cultures were rapidly chilled.

Then, cells were washed twice with ice-cold distilled water at least, and resuspended to

an OD600 of ~1.2 in glycin buffer (50 mM glycine-0.01% Tris X-100 buffer, pH8.0).

Samples were incubated at 37 with gentle agitation. Autolysis was detected every 30℃

min for 4 h in absorbance at OD600.

Transmission electron microscopy(TEM)Preparation and examination of S. epidermidis by electron microscopy was performed as

described previously by Cui et al[19]. Briefly, exponential phase cells were harvested,

fixed with 2.5% glutaraldehyde followed with postfixation in 1% osmium tetroxide and

then 1% aueous uranylacetate. Then, samples were embedded in Epon 812. The sections

were stained with uranyl acetate and lead citrate. Thirty cells of each strain were

measured for cell wall thickness using TEM(FEI Tecnai G2 20 Twin, FEI, USA), and the

results were expressed in the form of mean value+standard deviation (SD).

Analysis of clone correlation via Pulsed-field gel electrophoresis (PFGE)PFGE of SmaI-digested (TaKaRa, Japan) genomic DNA of Staphylococcus epidermidis

was performed using CHEF-Mapper XA PFGE system (Bio-Rad, CA, USA) for 23 hrs at

6 V/cm and 14 (Block 1: pulse times from 5 to 15 seconds for 10 hrs, Block 2: pulse℃

times from 15 to 60 seconds for 13 hrs). Strains showing same sizes and number of band

patterns were considered identical, and the strains varied with only two or three bands

were regarded as possibly clonally related.

Statistical analysisStatistical analysis was estimated by two-tailed Student's t test. A p value <0.05 was

considered statistically significant.

Results

Organisms and antimicrobial susceptibility characteristics12 non-duplicate teicoplanin non-susceptible S. epidermidis clinical isolates were

included in our study, which were isolated from cerebrospinal fluid(6 isolates), ascites (2

isolates), wound secretion(2 isolates), and blood (2 isolates) specimens. The mean age of

these patients was 42.5 years (range 19-64 years), seven males and five females were

included. Of note, two patients (16.7%) died of infections. The main characteristics of

these patients and their previous exposure to antimicrobial agents were summarized in

Table 1. All of 12 S. epidermidis were detected with a decreased susceptibility to

teicoplanin, according to the broth microdilution method and the E-test assay (Table 2),

consisting of 4 teicoplanin intermediate-resistant S. epidermidis and 8 teicoplanin

resistant S. epidermidis. Overall, all strains were methicillin resistant, showing several

multidrug-resistance patterns (Table 2), but remained susceptible to linezolid, tigecycline

and vancomycin. In addition, vancomycin susceptibility was further identified by

PAP/AUC, as shown in Fig 1, all of isolates showed heterogeneous resistant to

vancomycin.

Table 1. Characteristics of 12 teicoplanin non-susceptible S. epidermidis Isolate Sex Age

(years)Specimen Word Clinial diagnosis Antibiotic

exposuredaOutcome MLST SCCmec

typePFGE type

Sep-9 Female 49 Cerebrospinal fluid Neurosurgery Intracranial infection TEC, SCF, CLI, VAN

Death ST59 II D

Sep-28 Male 41 Cerebrospinal fluid Neurosurgery Intracranial infection TEC, SCF, CLI, VAN

discharge ST59 II D

Sep-42 Male 23 Cerebrospinal fluid Neurosurgery Hydrocephalus TEC,VAN discharge ST267 III ASep-143 Male 15 Cerebrospinal fluid Neurosurgery meningocephalitis TEC,VAN discharge ST267 III ASep-BL03 Female 64 Blood ICU Septicemia VAN Death ST267 III ASep-BL59 Male 36 Blood ICU Multiple injuries VAN discharge ST267 III ASep-547 Male 49 Secretion Burn unit Severe burn LEV, SXT, LNZ discharge ST328 III CSep-590 Male 62 Cerebrospinal fluid Neurology Intracranial infection TEC,VAN discharge ST267 III BSep-35 Female 59 Cerebrospinal fluid Neurology Intracranial infection TEC,VAN discharge ST267 III BSep-05 Female 46 Secretion Trauma surgery traumatic

pneumothoraxCefodizime discharge ST267 III A

Sep-73 Male 19 Ascites Infectious disease Cirrhosis Cefamandole, fusidic acid

discharge ST262 III C

Sep-138 Female 47 Ascites Gastrointestinal surgery

Pancreatic cancer TEC, SCF, CLI, VAN

discharge ST267 III A

aAbbreviations used: SCF, Cefoperazone/sulbactam; CLI, Clindamycin; LEV, Levofloxacin; LNZ, Linezolid; VAN, Vancomycin; TEC, Teicoplanin; SXT: Trimethoprim/sulfamethoxazole.Table 2.Antibiotic susceptibilities of 12 teicoplanin non-susceptible S. epidermidis(μg/ml)

Antimicrobial agentsa

Sep-9 Sep-28 Sep-42 Sep-143 Sep-BL03 Sep-BL59 Sep-547 Sep-590 Sep-35 Sep-05 Sep-73 Sep-138

OXA >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256PEN >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256CZO >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256CXM >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256FEP >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256FOX >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256ERY 16 64 >256 >256 16 >256 64 64 >256 >256 64 >256

CLI 4 16 4 >256 8 32 >256 >256 >256 >256 16 >256LEV 1 2 >256 >256 1 >256 >256 >256 >256 >256 1 0.5CIP 1 2 >256 >256 4 >256 >256 >256 >256 >256 0.5 1TOB 1 1 >256 >256 2 >256 >256 >256 >256 >256 2 4GEN 2 2 >256 >256 4 >256 >256 >256 >256 >256 2 4RIF 1 1 >256 >256 2 4 1 >256 >256 >256 1 1SXT 1/19 1/19 1/19 8/152 1/19 1/19 1/19 2/38 2/38 8/152 1/19 1/19LNZ 2 2 1 2 2 2 2 2 1 2 2 2TGC 0.5 0.5 0.5 0.5 0.25 0.5 0.5 0.5 0.25 0.5 0.5 0.5VAN 2 2 1 2 4 4 4 2 1 2 2 4TEC 16 32 32 32 16 128 32 32 16 128 16 64TEC-Etest 12 64 48 48 12 64 32 32 16 64 16 48

aAbbreviations used:OXA, Oxacillin; PEN, Penicillin; CZO, Cefazolin; CXM, Cefuroxime; FEP, Cefepime; FOX, Cefoxitin; ERY, Erythromycin; CLI, Clindamycin; LEV, Levofloxacin; CIP, Ciprofloxacin; TOB, Tobramycin; GEN, Gentamicin ; RIF, Rifampicin; SXT: Trimethoprim/sulfamethoxazole; LNZ, Linezolid; TGC, Tigecycline; VAN: Vancomycin; TEC, Teicoplanin.

Molecular epidemiologyTen out of the twelve teicoplanin non-susceptible S. epidermidis harboured the SCCmec

type III gene, SCCmec II was detected in other two isolates, all of these isolates displayed

mecA gene positive. None of those three genes, VanA, VanB and VanC were found.

MLST revealed four different sequence types among these isolates(Table 1): ST267(8

isolates), ST59(2 isolates), ST328(1 isolates), ST262(1 isolates), respectively. The two

isolates carried a SCCmec type II gene belonged to ST59. PFGE result showed that four

distinct pulsotypes were identified among the twelve isolates: type A (6 isolates), type B

(2 isolates), type C (2 stisolates), and type D(2 stisolates)(Table 1, Fig 2), six isolates

belonged to ST267 shared the same PFGE pattern( type A).

Susceptibility to Triton X-100 induced autolysisThe analysis of triton X-100 induced autolysis revealed a dramatic reduction in autolytic

activity for all of twelve isolates compared to teicoplanin-susceptible S. epidermidis

strains ATCC 12228 (Fig.3)

Analysis of transmission electron microscopy A total of 5 S. epidermidis strains, Sep-BL03, Sep-35(MIC of TEC=16μg/ml), Sep-

547(MIC of TEC=32μg/ml), Sep-BL59(MIC of TEC=128μg/ml), and a standard

teicoplanin-susceptible S. epidermidis strains ATCC12228 were subjected to

morphometric study using TEM. The result observed an increase in cell wall thickness of

all S. epidermidis strains. Furthermore, teicoplanin resistant S. epidermidis isolates had

significant thicker cell walls than teicoplanin-susceptible S. epidermidis strains ATCC

12228 and teicoplanin intermediate-resistant S. epidermidis(Sep-BL03 and Sep-35)

(Fig.4). Nevertheless, there was no significant difference between S. epidermidis strains

ATCC 12228 and teicoplanin intermediate-resistant S. epidermidis(Sep-BL03 and Sep-

35).

DiscussionDespite coagulase-negative staphylococci (CoNS) resistance to glycopeptide antibiotics

has been first reported in 1986[8], However, because of the genetic and biochemical

mechanisms of resistance to glycopeptide in enterococci were rapidly and exhaustively

elucidated, and the transfer of drug resistance genes easily lead to the spread of resistance

(VanA, VanB, and VanC gene), unlike the mechanisms of resistance in CoNS, major

interest remained centered on enterococci, Although, rare hospital acquired outbreaks

occurring with staphylococcal strains with decreased susceptibility to teicoplanin were

reported, data regarding CoNS species that glycopeptide resistant remain scarce for most

locations[20, 21]. In the present study, 12 teicoplanin non-susceptible S. epidermidis were

observed in The First Affiliated Hospital of Zhengzhou University from Jan 2013 to Oct

2013( including 4 teicoplanin intermediate-resistant S. epidermidis and 8 teicoplanin

resistant S. epidermidis.).

Long-term use of glycopeptide antibiotics is the major reason for the emergence of

glycopeptide resistant strains in some studies, glycopeptide antibiotics (vancomycin and

teicoplanin) are regarded as one of the mainstays of treatment for MRSA(Methicillin

resistant Staphylococcus aureus) and MRCoNS (Methicillin resistant coagulase-negative

Staphylococci) infections[22-24], the usage of vancomycin in our hospital is common,

which led to the appearance of teicoplanin non-susceptible S. epidermidis. Nine out of the

twelve teicoplanin non-susceptible S. epidermidis with glycopeptide drug exposure,

despite all clinical isolates showed susceptibility to vancomycin (MICs, 1-4μ g/mL),

PAP/AUC results showed profiles indicating heterogeneous resistance to vancomycin,

implying that teicoplanin resistance might be considered a screen marker for vancomycin

heteroresistance in staphylococci.

Furthermore, 12 teicoplanin non-susceptible S. epidermidis isolates were subjected to

SCCmec typing and mecA detection, the result demonstrated that all of these strains

carried the mecA gene, resulting in a quite high level of oxacillin and consequently β-

lactam resistance. Ten isolates were SCCmec type III, a predominant type in China,

which was linked with the previous report[25,26], the other two isolates were SCCmec

type II. By using MLST and PFGE method, we further analysed the molecular

characteristics of 12 S. epidermidis clinical isolates. The MLST result observed that the

most represented sequence type was ST267 for the first time, which comprised

66.67%(8/12) of all clinical isolates in the study, the other sequence types were ST59 (2

iaolates), ST328 (1 isolate) and ST262 (1 isolate) , respectively. In previous

epidemiological studies, ST2 has usually been considered to be the most prevalent ST

type among S. epidermidis strains worldwide, including China[13, 25, 27-29].

Conversely, our study presented an overall greater proportion of ST267 strains,

suggesting clonal spread of the teicoplanin non-susceptible S. epidermidis strains in this

region. It is the first report that the ST267 clone has spread in our hospital in China.

PFGE analysis also revealed spread of 1 predominant clonal type (clone A, 6 isolates ),

which appeared consistent with the possibility of a clonal expansion. Unfortunately, since

limited number of strains were obtained, the prevalence and spread of the ST267

teicoplanin non-susceptible S. epidermidis isolates in this region still need to be further

monitored.

At present, the mechanism of resistance to glycopeptide in staphylococci is still unclear,

Most of the studies have focused on vancomycin-resistant laboratory mutant of MRSA,

Common features of glycopeptides non-susceptible staphylococcus include cell wall-

thickening and reduced autolysis[10-12,30]. In the current study, TEM also showed

thickening of cell wall among these clinical strains, versus the standard strain ATCC

12228. Teicoplanin resistant S. epidermidis had significantly thicker cell walls than ATCC

12228 and teicoplanin intermediate-resistant S. epidermidis. Previous studies have shown

that the mechanism of resistance to glycopeptide was rather complicated, several changes

affecting cell wall metabolism and avoidance of glycopeptide molecules reaching the

sites of peptidoglycan synthesis were found, demonstrating that cell wall thickening is

responsible for both vancomycin and teicoplanin resistance[10-12]. Reduced autolysis

also been detected in these clinical isolates.

Taken together, our findings demonstrated the prevalence of S. epidermidis isolates with

reduced susceptibility to teicoplanin, and identified a potential endemic clone of ST267

among teicoplanin non-susceptible S. epidermidis isolates in this region, which was the

first report in China. The presence of sequence type 267 teicoplanin non-susceptible S.

epidermidis isolates highlights the importance of early detection and surveillance of

CoNS for resistance to glycopeptides, and strict control measure should be applied to

prevent their further dissemination.

Funding No funds were received for the realisation of this work

Conflict of interest The authors declare that they have no conflict of interest.

Ethical approval This article does not contain any studies with human participants or

animals performed by any of the authors

Informed consent Informed consent was obtained from all individual participants

included in the study

Open Access This article is distributed under the terms of the Creative Commons

Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/),

which permits unrestricted use, distribution, and reproduction in any medium, provided

you give appropriate credit to the original author(s) and the source, provide a link to the

Creative Commons license, and indicate if changes were made.

Figure legends:

Fig 1. PAP/AUC analysis of the representative strains and the control strains ATCC25923, Mu3 and Mu50. Each test was repeated 3 times.

Fig.2. Pulsed-field gel electrophoretogram results of the chromosomal DNA fragments.

Fig.3. Triton X-100 stimulated autolysis assay. The percentage of the remaining absorbance at OD600 nm of each strain on 30 min intervals was plotted. The test was repeated 3 times, and 1 representative was indicated.

Fig.4. Transmission electron microscopy analysis of the representative strains. (A) Transmission electron microscopy of ATCC 12228, Sep-BL03, Sep-35, Sep-547 and Sep-BL59. Magnification, ×25000. (B) Comparison of cell wall thickness between the strains ATCC 12228, Sep-BL03, Sep-35, Sep-547 and Sep-BL59. The data are expressed as the mean±SD of 30 cells of each strain for determination and were evaluated by the Student's t test, where a P value less than 0.05 were considered statistically significant. NS=not significant.References

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类别：临床微生物检验916436

19株弗兰西斯菌分子生物学鉴定及其系统分类学分析李工厂 王婷 何燕霞

郑州大学第二附属医院检验科 郑州 450014【摘要】： 目的 对19株弗朗西斯菌进行菌种鉴定和系统发育分析。方法 对环境水样中分离得到的19株弗朗西斯菌进行16S rRNA sdhA、mdh、rpoB基因进行测序分析，构建系统发育树进行分类学研究。结果 16S rRNA测序分析为Francisella

philomiragia分支包括 HN-17、HN-19、YJ-2、D3-1、HN-4、QH-5、QH-2、QH-

3、QH-12、QH-13 共10株分离株，其与参比菌株Francisella philomiragia 亲缘关极近，其相似性99.2%-99.5%之间。Francisella guangzhouensis 分支上由

YG23、10HP457、10HP1970、10HP82-10、10HL1958，共五株，相似性率为98.7%-98.8%；sdhA, mdh, rpoB基因扩增测序分析，菌株D3-2的 mdh基因、D-4 的mdh,sdhA基因和10HL1958的rpoB基因扩增失败。将余下17株分离株的持家基因（ sdhA, mdh, rpoB）的部分序列进行串接后进行系统发育分析，如图所示供试菌株与参比菌株共同组成系统发育树的三大分支：HZH-2独自形成一分支，与参比菌株的相似性分别为85.3%、87.8%；10HL457、10HP1970、10HP82-10、10HL1938四株菌连同两参比菌株共同构成发育树的分支，其相似性为98.5%-99.3%，余下11株分离共同组成发育树的最后一个分支，其与参比菌株蜃楼弗朗西斯菌的相似性较高为97.1%-97.8%。 结论 研究发现分离菌中11株与蜃楼弗朗西斯菌相似性最高而被鉴定为蜃楼弗朗西斯菌，1株为弗朗西斯菌科候选属，其余7株菌株被暂鉴定为疑似新种。研究中分离菌株蜃楼弗朗西斯菌为主导（57.8%,11/19），疑似新种（36.84%，7/19），在一定程度上表明我国弗朗西斯菌多样性程度较高，同时亦是国内首次大范围的对环境水中弗朗西斯菌多样性调查的研究。【关键词】弗朗西斯菌 16S rRNA 系统发育树

弗朗西斯菌（Francesella）隶属于-变形菌门的弗兰西斯科，为兼性细胞内寄生的革兰氏阴性苛养性细菌。现属内共包括6个有效命名种，模式菌种为强毒力致病菌土拉热弗兰西斯菌[1]，该菌为土拉热病的病原菌，现已被认为是一种潜在的生物恐怖武器，其种内细分为 F. tularensis subsp. tularensis, F. tularensis subsp.

holarctica, F. Tularensis subsp. novicida and F. tularensis subsp. mediasiatica 四个亚种，

常见的土拉热菌病大多由土拉热弗朗西斯菌的美洲亚变种和欧美变种引发，除此之外，近年来研究发现属内蜃楼弗朗西斯菌和西班牙弗朗西斯菌也可引发人类相关疾病[2]本研究对国内环境水中分离得到的19株弗朗西斯菌进行16S rRNA 和多个位点基因: 16S rRNA、sdhA[2]、mdh、rpoB 进行系统发育分析，旨在揭示我国环境水中弗朗西斯菌的多样性及其系统发育地位。1 对象和方法1.1 材料1.1.1供试菌株

菌株 水样来源 水样类别 分离年月 GenBank

QH-2 青海湖 咸水 2014.07 KU593485

QH-3 青海湖 咸水 2014.07 KU593486

QH-12 青海湖 咸水 2014.07 KU593489

QH-15 青海湖 咸水 2014.07 KU593490

HN-1 海南 海水 2014.07 -

HN-4 海南 海水 2014.07 -

HN-17 海南 海水 2014.07 -

HN-19 海南 海水 2014.07 -

D3-1 外伶仃岛 海水 2014.07 -

D3-2 外伶仃岛 海水 2014.07 -

D4 外伶仃岛 海水 2014.07 --

YJ-2 阳江 海水 2014.09 -

HZH-2 海珠湖 淡水 2014.07 -

10HL1958 广州市疾控中心 空调水 2010.10 JN620407

10HL1970 广州市疾控中心 空调水 2010.10 JN620409

10HP457 广州市疾控中心 空调水 2010.10 JN620411

10HP82-10 广州市疾控中心 空调水 2010.10 JN620410

10HL1938 广州市疾控中心 空调水 2010.10 JN620406

YG23 澳门 海水 2010.10 JN620412

。1.1.2 主要试剂

PCR试剂（TakaRa 公司），TaqTM 酶体系及 DL 2000 Marker（大连 TaKaRa

公司）Goldview核酸染料，Tris-碱，硼酸，Ezup 柱式细菌基因组DNA 抽提试剂盒（上海生工）；引物合成及测序交由上海英捷维基公司完成。1.1.3 主要仪器

ABI Veriti 96 孔热循环仪 (美国 ABI 公司)；DYY-6C 型电泳仪；全自动凝胶成像系统（英国 Syngene 公司）；Chelex提取液（实验室自配）；NanoDrop 2000 C

分光光度计（ThermoFisher Scientific）；MaxQ4000恒温摇床（广东丹利科技有限公司）；SW-CJ-IFD 型超净工作台（六一仪器厂，北京）；1.1.4溶液配制

（1）PCR反应体系配制，见表 2-1

表 2-1 PCR反应体系试剂 体积/ulTakaRa Taq(5U/ul) 0.510*PCR buffer(Mg2+Plus) 2

dNTP Mixture(2.5mM) 2

模板DNA 2

Primer 1 1

Primer 2 1

ddH2O Up to 20ul

1.2 方法1.2.1 菌株复苏室温解冻冻存菌株，选用高压灭菌吸头吸取 20ul 菌液，分区划线接种于

BCYEα基础培养基，37℃培养 48h，观察菌落生长情况。1.2.3 总DNA提取 供试菌株经 BCYEα 基础培养基活化后，挑取单个菌落接种，接种于 5 ml

BCYEα液体培养基，置于 35℃摇床内 250 r/min，摇床培养至对数生长期（2-

3d），收集菌悬液 1.5ml于 12000rpm/min离心 3min，弃去上清加入生理盐水洗涤细菌，于 12000rpm/min离心 3min，弃去上清，加入 500ul的 Chelex提取液，用移液器吹打混匀，100℃加热 15min后 12000rpm/min离心 1min，上清液即为模板DNA，-20℃冻存备用。

1.2.4 16S rRNA 序列分析16S rRNA PCR 扩增引物为细菌通用引物 27F、1492R ,引物序列见表 2-3。扩

增参数：94℃，预变性，5min； 35个循环（94℃，变性，30S；52℃，退火， 30

S；72℃，延伸，1 min）；72℃，延伸，7 min。PCR 产物经 0.8%琼脂糖凝胶电泳，凝胶成像确认后送上海英捷维基公司广州测序部进行基因测序。将测得序列使用 Blast 软件进行在线比对，并从 GenBank 数据库中下载与待分析序列相近的已知种模式菌株的 16S rRNA 序列。序列比对和系统发育的构建都采用 MEGA5.2软件完成。序列比对采用 ClustalW2.0，系统发育的构建采用邻接法 ( Neighbor-joining

method) 构建发育树，自展值( Bootstrap ) 为 1000，分支上树形可信度大于 50 的显示。1.2.5 持家基因（sdhA、mdh、rpoB）基因序列分析三种基因引物序列见表 2-3。（1）sdhA基因扩增参数：94℃，预变性，5 min；32个循环（94℃，变性，

30S；54℃，退火，30 S；72℃，延伸，1 min）；72℃，延伸，7 min。（2）mdh 基因扩增参数：94℃，预变性，5 min；32个循环（94℃，变性，

30S；58℃，退火，30 S；72℃，延伸，1 min）；72℃，延伸，7 min。（3）rpoB 基因扩增参数：94℃，预变性，5 min；32个循环（94℃，变性，

30S；54℃，退火，30 S；72℃，延伸，1 min）；72℃，延伸，7 min。PCR产物经凝胶成像确认后测序，对所得测序结果运用 DNAMAN 拼接获取

各个基因序列，构建各基因的系统发育树图，构建方法同 16S rDNA基因。表 2-3. 16S rDNA ,sdhA,mdh和 rpoB基因序列

Table.2-3 The sequence of 16S rDNA , sdhA, mdh and rpoB gene

引物名称 序列16S rDNA基因 27F 5 -AGAGTTTGATCMTGGCTCAG-3

1492R 5 -GGTTACCTTGTTACGACTT-3

sdhAsdhA-F 5-AAGATATATCAACGAGCKTTT-3

sdhA-R 5-AAAGCAAGACCCATACCATC-3

mdhmdh-F 5-GCT TRT TGG TGC TGG TAA TA-3

mdh-R 5-RCT TTC WGC CAT TTG RAT WC-3

rpoBrpoB-F 5-GAT GAT ATC GAT CAY CTD GG-3

rpoB-R 5-TTC VGG CGT TTC AAT NGG AC-3

2 结果2.1 16S rRNA基因序列分析 对供试菌株进行16S rRNA 基因扩增后均得到1500 bp 左右长度的片段，见图2-

1，经正反引物测序获得16S rRNA 基因正反序列，运用DNAMAN软件完成序列拼接， 将序列用Clustal-W 比对后剪齐得到1375bp 的序列运用MEGA5构建16S rRNA

系统发育树，见图2-2。如图所示，HZH-2和D3-2二者共同组成一个分支，其余17

株分离株与参比菌株Francisella philomiragia，Francisella

guangzhouensis，Caedibacter taeniospiralis构成三个分支。Francisella philomiragia

分支包括 HN-17、HN-19、YJ-2、D3-1、HN-4、QH-5、QH-2、QH-3、QH-

12、QH-13 共10株分离株，其与参比菌株Francisella philomiragia 亲缘关极近，其相似性在99.2%-99.5%之间。

图2-1 16S rRNA基因PCR扩增结果注： N,P分别为阴阳性对照； S1-19为待测样本； M为Marker Francisella guangzhouensis 分支上由 YG23、10HP457、10HP1970、10HP82-10、10HL1958，共五株，相似性率为98.7%-98.8%，相似性较低，暂怀疑其为新种。

图 2-2 19 株分离菌 16S rRNA 基因系统发育树Fig.2-2 Phylogenetic tree of 16S rRNA gene sequences of 19 isolates' ． The tree was

constructed by the Neighbor-Joining method ． Numbers at the nodes are bootstrap

percentages for 1000 samplings; values below 50% are not shown ． The scale bar indicates 2% nucleotide substitutions per site

2.2 持家基因（ sdhA, mdh, rpoB）基因序列分析依据 16S rRNA基因序列分析结果，对 19 株供试菌株添加 2 株参比菌株（广州

弗朗西斯菌和蜃楼弗朗西斯菌）共计 21 株菌进行 sdhA, mdh, rpoB基因扩增测序分

HN-19

HN-17

YJ-2

D3-1

HN-4

QH-5

QH-12

QH-3

QH-2

QH-15

Francisella sp. 10HL1958 JN620407

Francisella philomiragia strain ATCC 25015 AY928394

Francisella halioticida JF290376。 Francisella sp. FN252413

Francisella novicida AY928396F.tularensis Z21931

Francisella guangzhouensis FJ591095

Francisella sp. YG23 JN620412.

Francisella sp. 10HP457 JN620411Francisella sp. 10HP82-10 JN620410

Francisella sp. 10HL1970 JN620409Francisella sp. 10HL1938 JN620406

HZH-2

D3-2

Fangia hongkongensis AB176554 D-4

Caedibacter taeniospiralis AY102612Legionella pneumophila HQ287902.

8199

76

95

65

10092

99

100100

100

69

70

62

56

66

77

63

0.02

析，琼脂糖凝胶电泳发现菌 株 D3-2 的 mdh 基因、D-4 的 mdh,sdhA 基因和10HL1958的 rpoB基因扩增失败，暂无法参与构建系统发育树，具体原因还有待进一步研究。将余下 17 株分离株的持家基因（ sdhA, mdh, rpoB）的部分序列进行串接后进行系统发育分析，采用邻接法构建系统发育树，见图 2-3。如图所示供试菌株与参比菌株共同组成系统发育树的三大分支：HZH-2独自形成一分支①与参比菌 株的相似性分别为 85.3%、 87.8%； 10HL457、 10HP1970、 10HP82-

10、10HL1938四株菌连同两参比菌株共同构成发育树的分支②，其相似性分别为98.5%-99.3%；余下 11 株分离共同组成发育树的最后一个分支③，其与参比菌株蜃楼弗朗西斯菌的相似性较高为 97.1%-97.8%。经整体上分析发现持家基因 sdhA,

mdh和 rpoB基因序列拼接后的构建的系统发育树与 16S rRNA基因系统发育树基本一致，前者分析的离散程度相对来讲更高一些，但是又存在一定的差异，如蜃楼弗朗西斯菌在两树图的系统发育地位存在明显不同。

图 2-2 持家基因 sdhA, mdh和 rpoB基因序列拼接后的系统发育树

QH-3 QH-2 QH-12 QH-5 HN-19 YJ-2 HN-17 D3-1 HN-3 HN-4

QH-15 Francisella guangzhouensis

Francisella philomiragia Francisella sp. 10HP82-10 Francisella sp. 10HP457

Francisella sp. 10HL1970 Francisella sp. 10HL1938

HZH-2

67

6351

8399

100

9891

7395

56

87

0.2

①

②

③

Fig．2 Phylogenetic tree of concatenated sequences of sdhA, mdh and rpoB． The tree was constructed using the neighbor-joining method. Numbers at the nodes are bootstrap

percentages for 1，000 samplings; values below 50% are not shown．①,② and ③ are on behalf of three branches of the phylogenetic tree. The scale bar indicates 20% nucleotide substitutions per site.

3 结论 弗朗西斯菌与自然界中分布广泛，适应生存能力较强，属内土拉弗朗西斯菌、

蜃楼弗朗西斯菌、西班牙弗朗西斯菌与人疾病相关，临床症状多样，因菌种而异。土拉弗朗西斯菌致病力极强，早期主要为发热、头疼、寒战身体不适症状，该菌临床表现可分为：溃疡腺型、腺型、眼腺型、脑膜炎型、胃肠炎型、肺炎型、伤寒型[4]，以溃疡型最为常见。本研究采用16S rRNA基因、持家基因( sdh,rpoB 和 mdh)

系统发育分析等方法对分离自我国西北地区( 新疆、西藏、青海、内蒙古、宁夏、甘肃)、沿海省份（江苏、广东、广西、海南）和广州市内中央空调水等不同地点水样分离获得的19株黑弗朗西斯菌纯培养进行了系统分类学研究，水样类别涵盖了咸水、咸淡水、淡水和中央空调水，完成对我国环境水样中弗朗西斯菌分布与多样性的初步调查。目前，该研究亦是首次大范围的对我国环境水中弗朗西斯菌多样性调查的研究。对19株分离株进行16S rRNA和持家基因( sdh,rpoB 和 mdh)基因序列分析后发现分离菌中11株与蜃楼弗朗西斯菌相似性最高而被鉴定为蜃楼弗朗西斯菌，1株为弗朗西斯菌科候选属，其余7株菌株被暂鉴定为疑似新种。本次分离获得菌株蜃楼弗朗西斯菌为主导（57.8%,11/19），疑似新种（36.84%，7/19）的发现分离证明了王艳华等人认为我国弗朗西斯菌多样性程度令人吃惊的结论，同时警醒我国的弗朗西斯菌相关研究工作还处于起步阶段，迫切需要广大同仁共同努力，完善相关研究。结合菌株分离的水样来源和水质种类信息发现，蜃楼弗朗西斯菌属内对人致病

的病原菌，主要分布于海水，亦可在中央空调水中生长。此外研究发现现海陆两栖的弗朗西斯菌最初源自于海洋[2]，同时对土拉弗朗西斯菌的全基因组分析发现其毒力强弱与弗朗西斯毒力岛基因（Francisella Pathogenicity Island, FPI）的缺失和获得有关，综合以上信息研究认为蜃楼弗朗西斯菌可能存在着向强毒力、强致病性大规模感染人且栖息地由海洋经陆地至与人生活密切相关的空调水进化的趋势，介于水

样数量、地域的限制致使本研究存在一定的不足之处，同时本研究此次采用的实验室自制培养基是否适合土拉弗朗西斯菌生长因该菌的特殊性而未得到验证，进而致使本次研究过程中该菌未能检出。此外，该研究首次自淡水中成功分离获得疑似为弗朗西斯菌候选属内某菌菌株

HZH-2，菌株经纯化培养后进行保存，亦为目前国际上首次培养分离并且有菌株保存，进一步的深入研究还有待进行。 最后，本次研究分离菌株分离自环境水中或中央空调水，水与人类生活密切相关，同时分离获得疑似新种的毒力强弱、播散性等信息暂时还不清楚，尽管目前国内近有少数病例报道，但并不能排除弗朗西斯菌经水源性污染引起暴发流行的可能。参考文献[1] Pechous RD, McCarthy TR, Zahrt TC. Working toward the Future: Insights into

Francisella tularensis Pathogenesis and Vaccine Development. Microbiol Mol Biol R 2009;73:684.

[2] Goethert HK, Saviet B, Telford SR, 3rd. Metapopulation structure for perpetuation of Francisella tularensis tularensis. BMC microbiology 2009;9:147.

[3] Forslund AL, Salomonsson EN, Golovliov I, Kuoppa K, Michell S, Titball R, et al. The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensis. BMC microbiology 2010;10.

[4] Bandara AB, Champion AE, Wang XS, Berg G, Apicella MA, McLendon M, et al. Isolation and Mutagenesis of a Capsule-Like Complex (CLC) from Francisella tularensis, and Contribution of the CLC to F. tularensis Virulence in Mice. Plos One 2011;6.

类别：临床微生物检验842418 尿液分离的奇异变形杆菌耐药性及所产 β-酰胺酶分析

肖伟强＊ 沈勇 许青霞河南省肿瘤医院检验科，河南郑州

摘要 目的 研究尿液标本分离的奇异变形杆菌耐药性及所产 β-酰胺酶。方法 连续收集 2016年6月 1日至 2017年 6月 1日我院尿液标本共分离的奇异变形杆菌 31 株，PhoenixTM-100鉴定细菌种类，并测定最小抑菌浓度；双纸片协同实验测定超广谱 β-内酰胺酶的表型，PCR方法测定TEM, SHV, CTX-M, ACC, FOX, OXA, MOX, DHA, CMY1-23, ACT-1 and MIR等 β-内酰胺酶基因型；脉冲场凝胶电泳分析菌株菌株的同源性。 结果 亚胺培南和美罗培南的敏感率均为100%，其次哌拉西林/他唑巴坦、头孢西丁、阿莫西林/克拉维酸、头孢他啶、阿米卡星的敏感率分别为 98.59%、97.18%、94.37%、88.73%、85.92%，但对呋喃妥因、复方新诺明、氨苄西林、环丙沙星等敏感率均低于 50%；6 株头孢他啶和头孢噻肟耐药的奇异变形杆菌 ESBLs均为阳性；其中 4 株 ACC-1 型 β内酰胺酶阳性，3 株 CTX-M-1 型阳性，2 株 CTX-M-14阳性；3 株同时产 CTX-M-1和ACC-1的奇异变形杆菌具有典型的相关性。 结论 我院尿液分离的奇异变形杆菌可通过携带 CTX-M 型超广谱 β内酰胺酶或/和ACC-1 型 AmpC酶导致对头孢他啶和头孢噻肟耐药，部分菌株存在克隆传播的可能，因此合理使用抗生素，做好院内多重耐药菌的防控极其重要。 关键词 奇异变形杆菌；CTX-M；PFGE；ACC-1Analysis of the drug resistance and β-lactamases of Proteus mirabilis Isolates from urine samples XIAO Wei-qiang，SHEN Yong，XU Qing-xia＊. Clinical Laboratory, Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou 450000,ChinaCorresponding author：XIAO Wei-qiang. Email：xiaowq126@126.com【Abstract】 Objective To analysis the drug resistance and β-lactamases of Proteus mirabilis isolates from urine samples. Methods 31 Proteus mirabilis strains of urine samples were collected continuously from june 1st 2016 to june 1st 2017. The identification and the minimum inhibitory concentration (MICs) were determined by PhoenixTM-100；Extended Spectrum β-Lactamase（ESBLs）was analyzed by double-disk synergy test. The beta-lactamases genotype of TEM, SHV, CTX-M, ACC, FOX, OXA, MOX, DHA, CMY1-23, ACT-1 and MIR were performed by PCR. Pulsed-field gel electrophoresis (PFGE) was performed to analyze the homology of those isolates. Result The sensitive rates of imipenem and meropenem against Proteus mirabilis were 100%, and piperacillin/tazobactam, cefoxitin, amoxicillin/clavulanic acid, ceftazidime, amikacin were recorded to be 98.59%, 97.18%, 94.37%, 88.73%, 85.92%, respectively. The susceptibility rate to nitrofurantoin, sulfamethoxazole, ampicillin, ciprofloxacin

基金项目：河南省科技厅科技攻关项目（112102310256）作者单位：450000 郑州，郑州大学附属肿瘤医院检验科 *通讯作者：肖伟强，电子信箱：xiaowq126@126.com

were all less than 50%. Six isolates of Proteus mirabilis produced ESBLs, and they were all resisitant to ceftazidime and cefotaxime.Among these isolates, four isolates produced ACC-1-type AmpC beta-lactamases,three isolates produced CTX-M-1-type ESBLs, two isolates produced CTX-M-14-type ESBLs.Of particular note was that three solates were homology,and they produced CTX-M-1and ACC-1. Conclusions Some Proteus mirabilis isolates from urine samples in our hospital were resistant to ceftazidime and cefotaxime by encoding CTX-M-type ESBLs and/or ACC-1-type AmpC beta-lactamases. The results suggested that there exists cloning spread between different strains. So it

is very important to use antibiotics reasonably and to prevent and control the multidrug-resistant bacteria in our hospital.【key word】 Proteus mirabilis; CTX-M; PFGE; ACC-1奇异变形杆菌(Proteus mirabilis)是一种条件致病菌，广泛存在于人和动物的粪便、污水和土壤等自然环境中；同时奇异变形杆菌又是一种常见的临床病原菌，可引起血流、泌尿道和腹腔等部位的感染。随着抗生素在临床和农牧业的广泛使用，奇异变形杆菌对多种抗菌药物也表现出不同的耐药性，如国内外曾报道奇异变形杆菌携带多种 blaCTX-M 型超广谱 β-内酰胺酶、AmpC酶[1]和 blaOXA类多种碳青霉烯酶的报道[2]。不同国家、地区和医院，甚至同一患者不同的部位，其检出的细菌种类和耐药特点也不尽一致。为探明我院奇异变形杆菌的耐药表型及有无特殊的β内酰胺酶，并分析有无克隆株的流行，本文利用一年的时间，系统收集了我院所有尿液样本检出的奇异变形杆菌，现将研究结果报告如下。材料和方法仪器与试剂：细菌全自动鉴定和药敏分析仪购自美国 BD 公司，PCR 扩增仪购自美国ABI 公司，脉冲场凝胶电泳仪和凝胶成像系统购自美国 Bio-Rad 公司，PCR Pre-Mix、细菌总DNA提取试剂盒、合成引物等购自上海生工技术有限公司，E-test试纸条购自安图生物，部分药敏纸片购自OXOID 公司。菌株及来源：连续收集 2016年 6月 1日至 2017年 6月 1日来自我院各病区尿液标本共分离的奇异变形杆菌 31 株。尿液标本细菌的接种、培养、计数和鉴定均严格参考《全国临床检验操作规程》第四版， 质控菌株为大肠埃希氏菌 ATCC25922、沙门氏菌 H9812。菌株鉴定和药敏实验：所有奇异变形杆菌的鉴定和最小抑菌浓度测定均使用 PhoenixTM-100 完成，头孢他啶、头孢曲松、亚胺培南和美罗培南最小抑菌浓度的使用 E-test的方法验证，判读标准均参考 CLSI2016年 1月M100-S26。β-内酰胺酶表型和基因型确证实验：ESBLs表型确证实验参考 CLSI M100-S26。β-内酰胺酶基因型确证采用 PCR 扩增的方法，试剂盒提取菌株的基因组DNA作为模板，引物及扩增条件均参考相关文献，详见表 1。其余 β内酰胺酶基因blaTEM、blaSHV、blaFOX、blaOXA、blaCTX-M-2、blaCTX-M group 8/25、blaMOX、blaDHA、blaCMY1-23、blaACT-1和 blaMIR等扩增条件、引物均参考文献[6]。如扩增阳性，产物直接纯化后测序，结果在GenBank上比对。 脉冲场凝胶电泳及指纹图谱聚类分析：菌株在 LB肉汤中震荡过夜，菌液和 2%的低熔点胶灌模，使用 0.8g/L蛋白酶K消化 24小时，使用 TE洗胶及Xba 37Ⅰ ℃过夜酶切后，埋入 1%琼脂糖凝胶电泳，切换时间是 2.16-63.8秒，电场强度是 6V/cm，电场夹角是 120°，电泳时间是18.5h，最后用溴化乙锭染色紫外灯成像 ，判读参考 Tenover等人的标准[7]。表 1 本文所用的引物名称 引物 产物

长度 引物序列（ 5´-> 3´） 参考文献bla ACC AAC-F 346 CACCTCCAGCGACTTGTTAC [3]

AAC-R GTTAGCCAGCATCACGATCCblaCTX-M-1-like ALA2 941 ATGGTTAAAAAATCACTGCG [4]

P2D CAGCGCTTTTGCCGTGTAAGblaCTX_M-9-lik C-1 911 AACACGGATTGACCGTCTTG [5]

C-2 　 TTACAGCCCTTCGGCGAT 　结果

1. 菌株及药敏：共收集奇异变形杆菌 31 株。药敏结果显示，奇异变形杆菌对亚胺培南和美罗培南的敏感率均为 100%，其次哌拉西林/他唑巴坦、头孢西丁、阿莫西林/克拉维酸、头孢他啶、阿米卡星的敏感率分别为 98.59%、97.18%、94.37%、88.73%、85.92%，但对呋喃妥因、复方新诺明、氨苄西林、环丙沙星等敏感率均低于 50%。其中对头孢他啶和头孢噻肟耐药的 6 株奇异变形杆菌具体耐药谱见表 2。表 2 6 株奇异变形杆菌的最小抑菌浓度（μg/ml）

抗生素 DL0124 DL0135 BD0233 DL0347 BD0571 DL0829氨苄西林 2048 2048 2048 ≥2048 ≥2048 ≥2048阿莫西林/克拉维酸 32 32 4 16 64 >128替卡西林 >128 >128 >128 >128 >128 >128哌拉西林 >128 >128 >128 >128 >128 >128哌拉西林/他唑巴坦 8 8 <1 8 >128 >128头孢他啶 >32 >32 >32 >32 >32 >32头孢他啶/克拉维酸 0.5 <0,25 0.5 <0,25 <0,25 >16头孢噻肟 >8 >8 >8 >8 >8 >8头孢噻肟/克拉维酸 <0,5 <0,5 <0,5 <0,5 <0,5 >16头孢西丁 >32 8 8 >32 >32 4

氨曲南 >32 16 8 >32 >32 16亚胺培南 0.5 0.5 0.5 0.5 1 1美罗培南 <0,25 <0,25 0.5 <0,25 <0,25 <0,25阿米卡星 32 1 2 4 8 4庆大霉素 2 >32 1 >32 32 2妥布霉素 32 8 1 16 8 2环丙沙星 <0,062 <0,062 >8 >8 >8 <0,062左氧氟沙星 <0,125 <0,125 >8 >8 8 0.25复方新诺明 >128 1 >128 >128 >128 >128呋喃妥因 16 8 128 8 >128 >128

2. β-内酰胺酶表型和基因型确证实验： ESBLs表型实验证实，6 株奇异变形杆菌均为阳性。扩增显示，DL0135、BD0233、DL0347、DL0829在约 346bp处均有清晰条带，测序比对的结果证明，均为ACC-1（详见图 1），DNA 序列一致率均为 100%；DL0135、DL0347、DL0829在 941bp处有清晰条带，测序证实均为 CTX-M-1（详见图 2），DNA 序列一致率均为 100%；DL0124、BD0571在 911bp处有清晰条带，测序结果证实为 CTX-M-14（详见图 3）。其余均为阴性。

图 1 ACC-1基因 PCR 扩增电泳图（M为Marker）

图 2 CTX-M-1群基因 PCR 扩增电泳图（M为Marker）

图 3 CTX-M-9群基因 PCR 扩增电泳图（M为Marker）5. 同源性分析：DL0135、DL0347和DL0829三株细菌的条带的差异小于两条，所以认为此三株细菌具有同源性（详见图 4）。

图 4 Xbal酶切后的脉冲场凝胶电泳图 讨论CHINET数据统计显示[8]，最近 10年间，变形杆菌属的检出呈上升趋势，其中奇异变形杆菌的检出率从 2005年的 17.2%升高到 2014年的 39.8%，其中 ESBLs的检出率为 31.4%，我院尿液奇异变形杆菌 ESBLs的检出率为 19.3%（6/31），比全国的结果低。CHINET统计奇异变形杆菌对头孢唑林、甲氧苄啶-磺胺甲唑耐药率超过 50 %[8]，对头孢哌酮-舒巴坦、哌拉西林-他唑巴坦、头孢他啶、头孢西丁、阿米卡星、替加环素耐药率低于 10 %（1.6 %～7.4 %），对亚胺培南和美罗培南均保持了较高的敏感率。从本文的数据来看，我院尿路奇异变形对青霉素类、一代头孢、复方新诺明和喹诺酮类等敏感率较低，对三代头孢、含酶抑制剂和碳青霉烯类敏感率较高，此结果与全国数据基本一致。CTX-M类超广谱 β内酰胺酶在 20多种细菌中皆有检出，特别是大肠埃希菌、肺炎克雷伯菌和奇异变形杆菌[4,9]。CTX-M是一种质粒介导的头孢菌素酶，包含多个快速增长的亚群，具有临床重要的意义[9]。CTX-M基因在世界各地不同菌株均有检出，但不同地区流型的 ESBLs的基因型或亚型有一定差异。欧洲以 CTX-M-1群为主，亚洲洲国家以 CTX-M-9群为主[9]。CTX-M-14是 CTX-M-9群的一种，最近从尿液分离的肠杆菌科细菌中，CTX-M-14的检出占有很高的比例。国内有报道，11 株奇异变形中，一半以上 CTX-M-14阳性[1]。我院 6 株超广谱 β-内酰胺酶阳性菌株中，均为 CTX-M-1或 CTX-M-14。具体 CTX-M 各亚型在奇异变形杆菌中所占的比例需要更大的样本数据支持。除了 CTX-M 型超广谱 β内酰胺酶外，还发现了有三株细菌同时产ACC-1 型 AmpC酶。以往在奇异变形杆菌中发现的AmpC酶以 CMY 型为主，其次为DHA-1[1]，本文却连续在三株奇异变形杆菌里面均发现ACC-1 型 AmpC酶，较为罕见。ACC-1[10]属于质粒介导，可通过细菌基因重组或不同的菌株传递获得，是院内多重耐药感染控制的对象。通常AmpC酶可导致菌株对广谱 β内酰胺类如青霉素类、头霉素、氨曲南等耐药，对头孢吡肟敏感，对碳青霉烯类几乎无影响；但ACC-1是个例外，它不介导对头霉素类耐药。AmpC酶可联合其他的耐药机制，使菌

株的耐药表型表现的更为复杂。本文 6 株产ACC-1和 CTX-M-1的奇异变形杆菌，部分菌株对头霉素类的头孢西丁也表现为耐药，可能是多种 β内酰胺酶的协同作用，也可能存在其他的耐药机制，如外膜通透的变化，以上问题有待进一步研究。 同源性分析的结果显示，3 株同时产ACC-1和 CTX-M-14的奇异变形杆菌具有典型的同源性。追踪病历发现，此三株细菌均来自我院泌尿外科病区三位不同的患者，他们分别接受了膀胱肿瘤切除术、阴茎癌腹股沟淋巴结清扫术和直肠癌术后放疗，均有尿路插管，因此推测可能是我院传播的克隆株。总之，通过本文的研究发现，我院尿液分离的奇异变形杆菌对碳青霉烯类、氨基糖苷类和含酶抑制剂的 β内酰胺酶类抗生素仍保持较高的敏感性，但个别菌株可通过携带 CTX-M 型超广谱β内酰胺酶或ACC-1 型 AmpC酶导致对三代头孢耐药；尤为重要的是，手术后使用尿管的病人，存在院内奇异变形杆菌克隆传播的可能，因此合理使用抗生素，做好院内多重耐药菌的防控极其重要。参考文献[1] 吴伟元，陆坚，卢月梅 等. 深圳市人民医院产超广谱 β-内酰胺酶和／或AmpC 酶奇异变形杆菌的流行及其分子特征[J]. 中华微生物学和免疫学杂志.2014，06：423-430. [2] Girlich D, Bonnin RA, Bogaerts P, et al. Chromosomal Amplification of the blaOXA-58 Carbapenemase Gene in a Proteus mirabilis Clinical Isolate[J]. Antimicrob Agents Chemother. 2017 ,61(2): e01697-16. [3] Sharif NM, Sreedevi B, Chaitanya RK,et al. Beta-lactamase antimicrobial resistance in Klebsiella and Enterobacter species isolated from healthy and diarrheic dogs in Andhra Pradesh, India[J]. Vet World. 2017 ,10(8):950-954. [4] Tawfik AF, Alswailem AM, Shibl AM, et al. Prevalence and genetic characteristics of TEM, SHV, and CTX-M in clinical Klebsiella pneumoniae isolates from Saudi Arabia[J]. Microb Drug Resist. 2011,17(3):383-8. [5] Carattoli A. Resistance plasmid families in Enterobacteriaceae[J]. Antimicrob Agents Chemother.2009 ,53(6):2227-38. [6] Al-Agamy MH, El Mahdy TS, Shibl AM. Fecal Colonization with Extended-Spectrum Beta-Lactamase and AmpC-Producing Escherichia coli[J]. Biomed Res Int. 2016:3704150. [7] Tenover FC,Arbeit RD,Goering RV,et al.Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electhophoresis:criteria for bacterial strain typing［J］.Clin Microbiol,1995,33(9):2233-2239.[8] 李金，胡志东，汪复 等. 2005-2014年 CHINET变形杆菌属、沙雷菌属、枸橼酸杆菌属、摩根菌属及普罗威登菌属细菌耐药性监测[J].中国感染与化疗杂志. 2016，16（3）：284-293.[9] Zhao WH, Hu ZQ. Epidemiology and genetics of CTX-M extended-spectrum β-lactamases in Gram-negative bacteria[J]. Crit Rev Microbiol. 2013,39(1):79-101. [10] Jacoby GA. AmpC beta-lactamases[J]. Clin Microbiol Rev. 2009.22(1):161-82.

类别：临床微生物检验908536 表皮葡萄球菌 icaA/icaD基因与前列腺炎中弹性蛋白酶检测临床应用

涂学亮 王坤 黄河三门峡医院 河南三门峡 472000

[摘要]目的 研究前列腺液表皮葡萄球菌 icaA/icaD基因水平和弹性蛋白酶水平及其联合检测在临床中的应用。方法 采用 PCR方法检测表皮葡萄球菌 icaA/icaD基因水平，ELISA法检测前列腺液中性粒细胞弹性蛋白酶含量，研究表皮葡萄球菌 icaA/

icaD基因水平与弹性蛋白酶表达水平在前列腺炎的临床意义。结果 icaA基因的检出率为 19.63%（129/657），icaD基因的检出率为 21.30%（140/657）；ELISA结果显示 273例患者前列腺液中弹性蛋白酶的含量≤800 ng/ml，占 41.55%；63例患者前列腺液中弹性蛋白酶含量在 800-1000 ng/ml，占 9.59%；有 321例患者前列腺液中弹性蛋白酶含量超过 1000 ng/ml，占 48.86%； icaA基因阳性标本中，弹性蛋白酶水平≥1000ng/ml患者占 72.1%（93/129），icaD基因阳性标本中，弹性蛋白酶≥1000ng/ml患者占 72.86%（102/140）。结论 icaA/icaD与前列腺液中弹性蛋白酶可作为前列腺炎尤其是急性前列腺炎感染快速辅助诊断。[关键词] 表皮葡萄球菌；icaA/icaD；弹性蛋白酶；前列腺炎Clinical application of combined detection of Staphylococcus epidermidis icaA/icaD

gene and prostatitis elastase

[Abstract] Objective To study the clinical application of icaA/icaD gene and elastase in

prostatitis. Methods The icaA/icaD gene in Staphylococcus epidermidis was detected by

PCR ,and the neutrophil elastase content in prostatic fluid was detected by ELISA. The

clinical significance of icaA/icaD gene and elastase expression levels in prostatitis was

studied. Results The rate of icaA gene was 19.63% (129/657). The rate of icaD gene was

21.30% (140/657). The ELISA results showed that the elastase(800 ng/ml) in 273 patients

was 41.55%; elastase (800-1000 ng / ml ) accounted for 9.59%; elastase content of 321

cases is more than 1000 ng / ml, accounting for 48.86%; icaA gene (+), elastase

≥1000ng/ml patients accounted for 72.1% (93/129). Among icaD gene

(+),elastase≥1000ng/ml accounted for 72.86% (102/140). Conclusion Both icaA/icaD

and elastase in prostatic fluid can be used as auxiliary diagnosis of prostatitis, especially

acute prostatitis infection.

[Key words] Staphylococcus epidermidis; icaA/icaD; elastase; prostatitis

表皮葡萄球菌是人体皮肤和黏膜的正常菌群，致病力很低，很少引起感染。近年来，随着留置导管、人工瓣膜等医用介入材料大量使用，相关感染日益增多[1]，表皮葡萄球菌已成为医院感染中常见的条件致病菌。前列腺炎是男性生殖系统常见并较难治疗的疾病。其中细菌感染引起的占%，前期研究显示细菌感染引起的前列腺炎中表皮葡萄球菌在前列腺液中的检出率为 30%。然而，细菌性前列腺炎中表皮葡萄球菌的相关研究较少。已有研究显示中性粒细胞弹性蛋白酶是前列腺炎辅助诊断指标，可判断炎症的程度，与精液质量呈负相关而且可指导临床治疗方案的选择及预后判断[2]。有研究显示 icaA/icaD基因在表皮葡萄球菌致病过程中发挥着重要作用[3]。本研究主要检测表皮葡萄球菌 icaA/icaD基因表达情况和前列腺炎患者前列腺液中弹性蛋白酶表达水平，研究 icaA/icaD基因与弹性蛋白酶相关性及其检测在前列腺炎诊断、治疗和预后中的应用。1 研究对象与方法1.1研究对象 收集 2013～2015年黄河三门峡医院的 657例病人的临床资料、体格检查及前列腺液检查结果。前列腺炎症状包括：有下腹部、会阴部或者腰骸等部位疼痛，且排除其他原发

病，有不同程度的排尿刺激症状，包括尿频、尿急、尿痛、尿不尽、尿滴沥，有尿道口脓性分泌物或性功能障碍等临床症状超过 3个月；或前列腺液涂片检查显示有卵磷脂小体减少，白细胞计数大于 10个/HP，或有脓细胞的存在[4]。其中其他的原发病包括：有该部位其他原发疾病如前列腺癌、前列腺结石或明

显前列腺增生；邻近部位如直肠等部位的相关疾病；有泌尿生殖系统其他部位感染，如睾丸炎、附睾炎、尿路感染[5]；有类似症状如腰椎疾病或其他可以引起类似症状；有结构功能障碍如尿道狭窄等[6]。

1.2 标本收集 分离收集的自 2013～2015年黄河三门峡医院的前列腺液以及生殖道分泌培养标本的 657 株表皮葡萄球菌做为实验菌株（剔除重复菌株，即同一患者，重复送检）。2013～2015年分离到符合要求的实验菌株数目分别为 197 株、220 株、240 株，标准菌株为购自卫生部临检中心的金黄色葡萄球菌 ATCC25923。icaA/

icaD基因阳性菌株为浙江大学附属儿童医院周明明老师惠赠1.3菌种分离鉴定 657 株待测菌和质控菌分批次转至哥伦比亚血琼脂平板，三区划线，电热恒温培养箱中 37±1℃，孵育 18～24小时。按照《全国临检操作规程（第四版）》，将分批次转种获得的纯培养的待测菌和质控菌进行标准化处理。首先进行革兰染色，初步筛选待测菌株，待测菌株和金黄色葡萄球菌质控菌株染色结果为阳性，均为革兰染色阳性呈葡萄状的球菌。利用定量连续分液器将梅里埃配套的上机专用生理盐水 3ml注入专用的细菌比浊管中，使用细长的无菌棉拭子，选择适量单个菌落，紧贴比浊管的管壁在进行细致研磨，研磨均匀后，进行震荡混匀，此过程在涡旋振荡器上完成，借助比浊仪将待测菌的菌悬液的浓度调整为 0.5Mac，浓度为 1.5×108cfu/ml，GP鉴定卡安装完毕后进行上机操作，约 10小时左右使用配套软件查看鉴定结果，显示均为表皮葡萄球菌，且可信度均在 98%以上。1.4 icaA/icaD基因片段扩增 反应体系共 50µl，其中包括 10×buffer5µl 、引物各 1µl

、 Taq 酶 0.25µl (1.25u)、d NTP 4μl(0.2Mm)、模板 1µl，并且加超纯水至 50µl。每次均设阴性对照和阳性对照。扩增条件为 94℃预变性 5min；继以 35个循环，每一循环包括 94℃变性 1min，55℃退火 1min及 72℃延伸 1.5min；72℃终延伸 7min。同时设立阴阳对照一并检测。扩增产物于 4℃环境放置待用或送公司进行测序。

表 1 icaA/icaD 基因的 PCR 引物及扩增产物的大小[25][26]

引物 序列 大小icaA

上游引物：TCTCTTGCAGGAGCAATCAA188bp下游引物：TCAGGCACTAACATCCAGCA

icaD上游引物：ATGGTCAAGCCCAGACAGAG

198bp 下游引物：CGTGTTTTCAACATTTAATGCAA

1.5 中性粒细胞弹性蛋白酶的检测 采用 ELISA双抗体夹心的方法检测。将浓度为3000ng/ml的标准品冻干粉以用 1ml溶解液完全溶解，并标号为 a，静置十分钟；

取 4个 EP管编号为 b、c、d、e并分别加入 100μl溶解液，将溶解后标准品 a吸取100μl加入到 b并混匀，将混合后的 b吸取 100μl加入到 c，以此类推加到 e，使 a-e

管的标准品浓度分别为；3000 ng/ml、1500 ng/ml、750

ng/ml、375ng/ml、187.5ng/ml；将待测标本 10μl用样本稀释液 990μl 稀释 100 倍，低速离心机 3000r/min离心 10分钟，取上清待用；打开包被微孔板。a-e标准品、样本加样量均为 100μl/孔，用样本稀释液作为空白对照加入量同标准品和样本；

以孵育振荡器（350~400r/min）37℃孵育 30min。 弃去封板膜。以洗涤液注满微孔，约 300μl每次浸泡 40s，共洗板 4次，最后拍干。 每孔加酶结合物 100μl，再次以封板膜封板。以孵育振荡器（350~400r/min），37℃孵育 30min。 加显色底物 100ul/

孔，室温避光反应 15min。

每孔加终止液 50ul。15min内上酶标仪双波长 450/630nm比色，计算出精浆中弹性蛋白酶的浓度。1.7统计学分析 表皮葡萄球菌 icaA/icaD基因的携带情况和前列腺液中 NE的水平应用 SPSS17.0进行统计分析，P<0.05为差异有统计学意义。

2 结果2.1表皮葡萄球菌 icaA/icaD基因表达情况 分离 657 株表皮葡萄球菌共检出 icaA阳性菌 株 129 株，阳性率 19.63%（129/657）， icaD 阳性菌 株 140 株，阳性率21.31%（140/657）。有研究显示表皮葡萄球菌的 icaA/icaD基因参与生物被膜的形成，在致病过程中发挥着重要作用。

图 1 表皮葡萄球菌 ica A、ica D基因的 PCR 扩增产物电泳图：M：250bp Marker；1：ica

A(188bp)； 3：ica D(198bp)； 2、4：阴性对照

2.2 前列腺液中中性粒细胞弹性蛋白酶的表达情况 ELSIA结果显示 273例患者前列腺液中NE的含量≤800 ng/ml，占 41.55%；63例患者前列腺液中NE含量在 800-

1000 ng/ml，占 9.59%；有 321例患者前列腺液中NE含量超过 1000 ng/ml，占48.86%（表 3和图 2）。结果显示弹性蛋白酶在表皮葡萄球菌引起的前列腺炎有不同程度的表达。

表 3.657份标本的NE的水平分布（NE单位：ng/ml）NE水平 NE≤800 800＜NE＜1000 NE≥1000

例数 273 63 321

图 2 前列腺液中NE水平分布2.3 表皮葡萄球菌 icaA/icaD基因与 NE水平联合检测 如表 4和图 3所示，比

较各组 icaA、icaD的基因携带情况，NE≤800 ng/ml组携带 icaA和 icaD的例数分别为 23和 20例，分别占该组 8.4%和 7.3%。而 NE800-1000ng/ml组携带 icaA的例数和 icaD的例数分别为 13和 18例，分别占比 20.63%和 28.57%。经 χ2检验统计分析，icaA和 icaD携带情况无统计学差异（P>0.05）。

NE≥1000ng/ml 组携带 icaA 和 icaD 的例数 93 和 102，分别占比 28.97%和31.7%，与前两组相比较，有统计学差异（P<0.05）。如图 4所示携带 icaA基因的表皮葡萄球菌感染者，NE≥1000ng/ml的患者占 72.1%（93/129），携带 icaD基因的表皮葡萄球菌感染者，NE≥1000ng/ml的患者占 72.86%（102/140）。

表 4. 表皮葡萄球菌 icaA/icaD基因与NE水平（NE单位：ng/ml）基因 NE≤800 800＜NE＜1000 NE≥1000

icaA 23 13 93

icaD 20 18 102

图 3 不同NE水平的 icaA/icaD基因携带情况

图 4 携带 icaA/icaD基因的NE水平

3.讨论表皮葡萄球菌是凝固酶阴性的葡萄球菌，目前是院内感染的重要菌群，感染

率逐年上升[7]。有研究显示 icaA和 icaD参与细胞间黏附素 PIA的合成， 在生物被膜中有重要作用[8]，PIA还在免疫逃逸中发挥重要作用，PIA是表皮葡萄球菌最主要的血细胞凝集素，可以减弱人类中性粒细胞的杀伤作用。PIA还与细菌毒力密切相关，已经在小鼠，大鼠和兔子等动物试验中得到证实[9]。icaA和 icaD在表皮葡萄球菌中发挥着重要作用。前列腺炎分为急性细菌性前列腺炎、慢性细菌性前列腺炎、急性非细菌性前列

腺炎和慢性非细菌性前列腺炎[10,11]。区分上述四种类型的主要实验室检查手段有前列腺液常规、细菌培养等，但是由于前列腺液本身的性状比较粘稠，涂片中白细胞不均匀，且易与其他细胞混淆，难以区分，加上受操作者专业水平的影响，使得前列腺液常规检查难以标准化，并且白细胞在炎症应答过程中可大量被破坏，从而造成漏检，而用 ELISA方法检测弹性蛋白酶则可以规避上述缺陷，客观的反映前列腺的炎症状态[12]。弹性蛋白酶主要存在于中性粒细胞和单核巨噬细胞中，是以水解弹性蛋白酶为特征的一类酶，在机体很多器官中也有不同程度的存在，中性粒细胞弹性蛋白酶（NE）是存在于中性粒细胞中的弹性蛋白酶。当致病微生物存在时，中性粒细胞会释放出释放 NE，消灭和溶解致病因素，从清除病原菌[13]。文中显示不同患者的弹性蛋白酶水平不同，有研究显示弹性蛋白酶可作为诊断，判断感染严重程度及预后的辅助指标，为临床医师提供指导[14]

不同患者前列腺液 NE含量与 icaA、icaD携带情况的结果显示，NE≤800组与800＜NE＜1000组 icaA和 icaD的携带率无统计学差异 (P>0.05)，而与NE≥1000组icaA和 icaD的携带率有统计学差异(P<0.05)。此外分析显示携带 icaA基因的表皮葡萄球菌感染者，其 NE≥1000ng/ml患者占 72.10%（93/129），携带 icaD基因的表皮葡萄球菌感染者，NE≥1000ng/ml患者占 72.86%（102/140），结果显示携带icaA、icaD基因的表皮葡萄球菌引起的炎症更加严重即 NE≥1000ng/ml，该检测为临床制定治疗方案提供一定的指导意义。【参考文献】[1] 张杰;李爱林; 彭少华等·428例慢性前列腺炎病原学检查及其临床意义[J]·中华医院感染学杂志 .2004，14(02):166-168

[2]曾东彪. 弹性蛋白酶浓度在ⅢA 型前列腺炎患者精浆中的变化及对精液影响研究[D].南华大学,2014.

[3] Shusheng Zhou, Xiaoguang Chao, Mingming Fei et al. Analysis of S. Epidermidis icaA and icaD

genes by polymerase chain reaction and slime production: a case control study[J] BMC

Infectious Diseases 2013, 13:242

[4] 王小燕，陈颖，黄云超等•表皮葡萄球菌生物膜形成相关基因在表皮葡萄球菌和白假丝酵母菌混合生物膜形成中的作用研究[J]•中国修复重建外科杂志，2015，29（1）:63-68

[5] 雷玉洁，黄云超，杨丽等•肺癌患者中心静脉导管相关表皮葡萄球菌 icaA、icaD表达及转化生长因子 β1与生物膜的关系[J]•中国组织工程研究 .2012;16(12): 2158-2162

[6] 邢铭友，刘莉娜，宋世会等•表皮葡萄球菌 ica操纵子与细胞间多糖粘附素及生物膜表型的相关性[J]•华中科技大学学报（医学版），2008，37(4): 449-452

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[14]王宝龙,杨长海,李黎明.前列腺液中性粒细胞弹性蛋白酶测定在慢性前列腺炎诊断中的意义[J].中国男科学杂志,2008(08):28-30.

类别：临床免疫检验872117Worse liver damages in HCV/HIV coinfection than HCV monoinfection: linking to

HIV-related oxidative stress

Xiang-Bo Huang1, Liang Ming1

1 Department of Clinical Laboratory, the First Affiliated Hospital of Zhengzhou

University, Zhengzhou, Henan, China.

Abstract

Background

HIV infection aggravates the progression of HCV liver damage in HIV/HCV-coinfected

patients and the underlying pathogenesis is multifactorial. Although higher oxidative

stress was frequently delineated in HIV or HCV monoinfection, the status of oxidative

stress in HIV/HCV coinfection and its contribution to HCV liver damage are lack of

understanding.

Methods

A total of 363 HBsAg negative and anti-HCV positive FBDs from a village of central

China were recruited in July 2005. Mortality rates of HCV monoinfection and HIV/HCV

dual infection from 2005 to 2009 were calculated and compared. In 2009, a total of 282

anti-HCV positive subjects were follow-up visited, which included 102 HIVneg chronic

HCV carriers, 76 HIVpos chronic HCV carriers, 56 HIVneg HCV resolvers, and 48 HIVpos

HCV resolvers. 18 healthy adults from the same village were used as controls. The

indexes of oxidative stress, including total glutathione (tGSH), oxidized glutathione

(GSSG), reduced GSH, malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and

zinc, were analyzed. In order to determine the stages of liver disease and the influence of

HIV infection on HCV liver damage, Liver ultrasound examination, APRI and FIB-4

indexes were calculated or performed respectively.

Results

Our results showed that higher end-stage liver disease (ESLD)-related deaths in

HIV/HCV coinfection than HCV monoinfection was found (p=0.027). Further, Poorer

liver ultrasound manifestation were presented in HIVpos subjects than HIVneg individuals,

both in chronic HCV carriers and HCV resolvers (p<0.001). HIVpos patients had higher

levels of serum tGSH (p<0.001), MDA (p<0.001), GSH-Px (p<0.05), GSSG (p<0.001)

and reduced GSH (p<0.001) than HIVneg individuals, both in chronic HCV carriers and

HCV resolvers. HIV coinfection significantly decreased serum zinc levels in chronic

HCV infected subjects (p=0.017) and HCV resolved subjects (p=0.012). HIV-infected

patients with higher ALT/AST have higher serum GSSG levels than those with normal

ALT/AST (p<0.05). Also, HIV infection significantly increases APRI (p=0.008 for

chronic HCV group; p=0.002 for resolved HCV group) and FIB-4 (p=0.014 for chronic

HCV group; p=0.027 for resolved HCV group) indexes. Importantly, serum level of

GSSG was positively correlated with APRI and FIB-4 indexes in HIV positive

individuals (p<0.001 for both). Meanwhile, the worse liver ultrasound manifestation, the

higher serum GSSG were found in chronic HCV carriers, either with HCV monoinfection

(p<0.001) or HIV coinfection (p=0.01).

Conclusions

Our study indicated that HIV infection accelerated HCV liver damages in chronic

HIV/HCV-coinfected patients. The increased oxidative stress mainly induced by HIV

coinfection was involved in aggravated liver damage in HIV/HCV-coinfected patients.

Keywords:

Oxidative stress, HCV, HIV, coinfection, oxidized glutathione, liver fibrosis

INTRODUCTION

Acute hepatitis C virus (HCV) infection usually develops into chronic phase in

the majority of cases. However, there are still approximately one quarter to one third of

HCV-infected subjects ending up with HCV spontaneous clearance according to

observations from different cohorts. It is well-known that HCV and human

immunodeficiency virus-1 (HIV-1) share similar transmission routes, most commonly

through virus-contaminated blood or blood products. In several villages in central China,

higher prevalence of HCV monoinfection and HIV/HCV coinfection were found in

villagers with unsanitary blood donation history occurred in the1990's3-6. More than 90%

of HIVpos subjects were positive for anti-HCV responses in these former blood donors

(FBDs). The cohort shared similar race, dietary patterns, living environment and virus

genotypes provided an excellent object for study of HIV/HCV coinfection.

The reciprocal interaction between HIV and HCV may alter the outcomes of

antiviral therapy and influence prognosis of HIV/HCV-coinfected patients. Aggravation

of HCV-related liver damage by HIV coinfection was supported by presence of higher

HCV viremia in HIV/HCV-coinfected patients7. The mechanisms included loss of CD4+

T helper cells and neutralizing antibodies9, dysfunction of dentritic cells (DCs)10, iron

overload11 , and oxidative stress might also be involved in.

Glutathione (GSH) is a tripeptide with a gamma peptide linkage between

the carboxyl group of the glutamate side-chain and the amine group of cysteine, serving

as a key antioxidant which prevents damages to important cellular components caused

by reactive oxygen species, such as free radicals and peroxides12. Total GSH exists in

both reduced GSH and oxidized state, glutathione disulfide (GSSG). Reduced glutathione

reduces disulfide bonds formed within cytoplasmic proteins to cysteines by serving as

an electron donor and synchronously reduced GSH is converted to its oxidized

form, GSSG13. In addition, as a component of the GSH cycle, glutathione peroxidase

(GSH-Px) is one of the most important antioxidant enzymes in humans. GSH-Px

catalyzes the breakdown of lipid peroxides and hydroperoxides in both extracellular and

intracellular compartments and protects integrity of membrane against peroxidative

interference and damage. Malondialdehyde (MDA)15 is also an important biomarker for

measurement of oxidative stress, which generates from lipid peroxidation of

polyunsaturated fatty acids and is cytotoxic due to its capability to induce

macromolecular crosslinking polymerization.

Increased status of oxidative stress was identified in both HIV16 and HCV17

infection in in vitro studies, though their cell tropisms were totally distinct. Long-term

oxidative stress led to overabundant free radicals in liver and contributed to liver fibrosis,

cirrhosis and carcinogenesis. Baum18 and Shin16 investigated the oxidative stress and

antioxidant status in HIV/HCV coinfection and HIV monoinfection and concluded that

increased oxidative stress and decreased serum antioxidants were present in HIV/HCV

coinfection compared to HIV monoinfection.

Although both HCV monoinfection and HIV/HCV coinfection induced higher

oxidative stress, information on the influence of HIV-induced oxidative stress on HCV-

related liver damage is limited. In this study, we selected serum oxidant and antioxidant

indicators as targets to investigate the possible relationship of higher circulating free

radicals in HIV/HCV-coinfected patients and aggressive liver damage. The results will

provide information for evaluating the different influences of HIV and HCV on host

status of oxidative stress and reveal the potential role of aggressive oxidative stress

induced by HIV infection on liver damage in the setting of HIV/HCV coinfection.

METHODS

Study cohort and clinical evaluation

A total of 363 HBsAg negative and anti-HCV positive FBDs from a village of

central China were recruited in July 2005. The cohort was followed up in July 2009 and

282 patients were successfully visited and serum samples were collected. 36 persons died

of opportunistic infection or end-stage liver disease or other causes and the remaining 45

persons were failed to visit due to loss of contact. Anti-HCV antibody, HCV RNA, anti-

HIV antibody and HIV RNA were detected to define the status of HIV/HCV infection.

HIV infection was defined by positive anti-HIV responses. Chronic HCV infection was

identified by positive anti-HCV response and positive results for HCV-RNA detection.

HCV spontaneous recovery was identified by both negative HCV RNA and detectable

anti-HCV response and confirmed by continuous negative for HCV RNA detection 6

months after initial detection. According to the status of HCV and HIV infection, 282

participants were divided into 4 groups, including 102 HIVneg chronic HCV carriers, 76

HIVpos Chronic HCV carriers, 56 HIVneg HCV resolvers and 48 HIVpos HCV resolvers. A

flow diagram for recruited persons is illustrated in Figure 1. All participants never

received any forms of HCV specific antiviral therapy. Inversely, all HIV positive subjects

had received regular or intermittent highly active antiretroviral therapy (HAART),

consisting of two nucleoside reverse transcriptase inhibitors (NRTIs): azidothymidine

plus didanosine, or stavudine plus lamivudine, and one non-nucleoside reverse

transcriptase inhibitor (NNRTIs), nevirapine. Besides, 18 age- and gender-matched

healthy individuals with negative HIV, HBV, and HCV infection were used as healthy

controls. All participants were interviewed by trained and qualified staff using a

standardized questionnaire, including detailed general information, blood donation

history, and usage of antiviral or antiretroviral drugs. The demographic and clinical

characteristics of all participants were shown in Table 1. The study was approved by the

institutional review authorities of Peking University Health Science Center (Approval ID:

PKUPHLL20090011). All patients provided written informed consent before enrollment

in the study.

Serum biochemical test and blood routine test were performed for each subject.

The liver function indexes, alanine aminotransferase (ALT), aspartate aminotransferase

(AST), total protein, albumin and bilirubin, were measured by traditional clinical

standardized methods on Unicer Dxc 800 Synchron Clinical System (Beckman Coulter,

Fullerton, CA, USA).

Liver ultrasound examination

Fasting liver ultrasound examination was performed on 282 participants

using a Convex Ultrasound Scanner (GE LOGIQ 9, GE Medical System, USA). Grey-

scale images of the liver were obtained using 3.5-5 MHz multifrequency transducer. All

ultrasonography were performed by two senior doctors with more than 10 years of

experience and ultrasound manifestations were divided into normal, altered

echostructure, hepatomegaly, diffuse liver parechyma lesions and fatty liver in our study.

Altered echostructure refers to intrahepatic hyperechoic and heterogeneous echotexture

evaluated by ultrasound with exception of diffuse liver parechyma lesions. Fatty liver and

its severity were diagnosed according to the criteria from the American Gastroenterology

Association including (1) a diffuse hyperechoic echotexture, (2) increased liver

echotexture compared with the kidneys, (3) vascular blurring, and (4) deep attenuation19.

HIV/HCV seropositive screening and confirmation

Plasma HCV antibody was detected using the Abbott Architect anti-HCV assay

(Abbott GmbH & Co KG, Wiesbaden, Germany) and confirmed by HCV-RIBA assay

(Wantai Biological Pharmacy). HIV infection was screened by HIV antibodies ELISA

assay (GBI Biotech Co., Ltd., Beijing, China) and confirmed by HIV Blot 2.2 WB assay

(HIV Blot 2.2 WB; Genelabs Diagnostics, Singapore).

HCV-RNA, HIV-RNA quantification and CD4+/CD8+ T-cell counts

Plasma HCV-RNA quantification were performed using the Abbott Real-Time

HCV Amplification Kit (Abbott Molecular Inc. Des Plaines, IL, USA) according to the

manufacturer’s instructions. The detection limit is 30 IU/ml. The plasma levels of HIV-1

RNA were measured with the Standard Amplicor HIV Monitor assay, version 2.0 (Roche

Diagnostics, Indianapolis, IN, USA), according to the manufacturer’s protocols. The

lower limit of detection was 40 copies/ml.

The CD4+ T-cell counts were measured by subjecting EDTA-treated whole blood

stained for CD3/CD4/CD8/CD45 in TruCount tubes to analysis with a FACSCalibur flow

cytometer (Becton Dickinson, San Jose, CA, USA). The absolute numbers of CD4+ T

lymphocytes were analyzed using MultiSET software (BD Bioscience, San Jose, CA,

USA).

Oxidative stress measurement

GSH colorimetric detection kit was purchased from Arbor Assays (Ann Arbor,

MI). Three types of glutathione (total, reduced and oxidized forms) were measured

according to the manufacturer’s instructions after treatment with 2-vinylpyridine. The

levels of serum GSH-Px and MDA were measured by the colorimetric detection kit

(Jiancheng,Nanjing, China) according to the manufacturer’s instructions. Serum trace

element zinc was determined by the deproteinization method using a Perkin-Elmer 503

atomic absorption spectrophotometer.

Liver Fibrosis stages

In order to evaluate the stages of liver disease and liver fibrosis, two values of the

noninvasive fibrosis models, the aspartate aminotransferase to platelet ratio index (APRI)

and the fibrosis index based on the four factors (FIB-4) score, were examined. FIB-4

index was calculated by a formula based on age, aspartate aminotransferase (AST),

alanine aminotransferase (ALT) and platelet count that in accordance with the stages of

liver fibrosis in viral hepatitis.

Statistical analysis

All statistical analyses were performed using GraphPad Prism for Windows,

version 5.0 (GraphPad Software Inc., San Diego, CA). Comparisons between groups

were conducted using unpaired t tests or Mann-Whitney U-tests. Pearson Chi-squared test

was applied for comparing ALT/AST values, HIV characteristics and HCV-related

mortality rates. Correlation analysis between serum GSSG and APRI or FIB-4 or

ALT/AST was evaluated by spearman correlation coefficient. All p-values were two-

tailed, and were considered significant when lower than 0.05.

RESULTS

Mortality rates analysis from 2005 to 2009

Among the 363 subjects recruited in 2005, 36 deaths were observed in 2009,

separated by 7 deaths in 223 HCV monoinfection and 29 deaths in 140 HIV/HCV

coinfected patients. Causes of deaths were presented in Table 2. A significant difference

(p<0.001) of total mortality was presented between HCV monoinfection (3.14%) and

HIV/HCV coinfection (20.7%). Importantly, end-stage liver disease (ESLD) was

responsible for 1/7 (14.3%) of non-AIDS related deaths in HCV moninfection, which was

significantly lower than that (8/12, 66.7%) in HIV/HCV coinfection (p=0.027).

Figure 1. A flow diagram for recruited persons in the study.

Poorer liver ultrasound manifestation were presented in HIV positive subjects than

HIV negative individuals, both in chronic HCV carriers and HCV resolvers

282 participants, who were successfully followed up in 2009, received a liver

ultrasound examination for evaluating the extent of liver injury. As shown in table 3,

poorer ultrasound manifestation was found in HIV positive subjects than HIV negative

individuals, both in chronic HCV carriers and HCV resolvers (both p<0.001). Altered

echostructure was observed in 26.5% of HIVneg chronic HCV-infected patients, while

28.9% were appeared in HIVpos chronic HCV-infected patients. Specially, higher

percentage of diffuse liver parenchyma lesions was found in HIVpos chronic HCV-

infected patients than in the HIVneg counterparts (39.5% vs. 2.0%). Also, in HCV resolved

population, higher frequency of both altered echostructure (54.2% vs. 28.6%) and diffuse

liver parenchyma lesions (12.5% vs. 0%) were found in HIVpos patients than in HIVneg

patients. No differences in the frequencies of hepatomegaly and fatty liver were observed

between HIVneg individuals and HIVpos individuals, either in chronic HCV carriers or

HCV resolvers.

Higher serum tGSH, MDA, GSH-Px and lower zinc concentrations were found in

HIV positive subjects than HIV negative individuals, both in chronic HCV carriers

and HCV resolvers

To compare the oxidative stress levels in five groups with different HIV/HCV

infection status, serum tGSH, MDA, GSH-Px and zinc concentrations were detected and

analyzed (Figure 2). Overall, HIV-infected patients, including HIVpos chronic HCV

carriers and HIVpos HCV resolvers, had higher concentration of MDA (chronic HCV:

p=0.042; resolved HCV: p=0.022), total GSH (chronic HCV: p<0.001; resolved HCV:

p<0.001) and GSH-Px (chronic HCV: p=0.036; resolved HCV: p=0.040) than healthy

controls. Moreover, higher levels of serumtGSH (p<0.001), MDA (p<0.001), GSH-Px

(p=0.036) were found in HIVpos chronic HCV carriers than HIVneg chronic HCV carriers.

Similarly, same trend was found between HIVpos HCV resolvers and HIVneg HCV

resolvers (tGSH: p<0.001; MDA: p<0.001; GSH-Px: p=0.045) (Figure 2A, B and C).

Unlike in HIV infection, no differences in the level of serum tGSH, MDA and GSH-Px

were found between chronic HCV infection and HCV spontaneous recovery, neither in

the presence of HIV coinfection or not. In addition, HIV infection was found to decrease

the concentration of serum zinc since lower levels of serum zinc were found in HIVpos

patients compared to HIVneg subjects in both chronic HCV carriers (p=0.017) or HCV

resolvers (p=0.012). Besides, statistical difference was found in serum zinc concentration

bewteen HIVneg chronic HCV carriers and HIVneg HCV resolvers (Figure 2D).

Figure 2. The comparison of serum MDA (A), total GSH (B), GSH-Px (C) and zinc (D)

concentration among 102 HIVneg chronic HCV carriers (●), 76 HIVpos Chronic HCV

carriers (○), 56 HIVneg HCV resolvers (■), 48 HIVpos HCV resolvers(□) and 18 healthy

controls(◇). Higher serum tGSH, MDA, GSH-Px and lower zinc concentrations were

found in HIV positive subjects than HIV negative individuals, both in chronic HCV

carriers and HCV resolvers. Median value for each group was indicated as red line.

Comparisons between groups were conducted using unpaired t tests or Mann-Whitney U-

tests. All p-values were two-tailed, and were considered significant when lower than

0.05.

HIV-infected patients with higher ALT/AST have higher serum GSSG levels than

those with normal ALT/AST

Though an increased level of serum AST (p<0.001) and a nearly increased level

of serum ALT (p=0.060) were found in HIVpos HCV resolvers in comparison with HIVneg

HCV resolvers (Figure 4A), no significant correlation between serum ALT or AST and

serum GSSG level were found either in HCV-monoinfected patients or HIV/HCV-

coinfected patients (Figure 4A).

Figure 3. The comparison of serum GSSG (A) and reduced GSH (B) concentrations

among HIVneg chronic HCV carriers (●), HIVpos Chronic HCV carriers (○), HIVneg HCV

resolvers (■), HIVpos HCV resolvers(□) and healthy controls(◇). Median value for each

group was indicated as red line. Comparisons between groups were conducted using

unpaired t tests or Mann-Whitney U-tests. All p-values were two-tailed, and were

considered significant when lower than 0.05.

.DISCUSSIONMDA is a classic marker of oxidative stress and frequently reported to be elevated

in liver and peripheral blood of HCV and HIV22 infected patients. In the present study, we

found that serum MDA were significantly increased in HIV infection compared with

HCV infection and healthy controls. However, no differences were found between HCV

groups and healthy controls. These data indicated that HIV infection, but not infection,

enhanced MDA production. As antioxidant parameters, both reduced GSH and

glutathione peroxidase (GSH-Px), were found elevated in HIV infection but not in HCV

infection, which was opposite to our preconception and also different from previous data

detected from erythrocytes of HCV-infected patients by Moustafa et al20. The discrepancy

might contribute to different sample type, erythrocytes or serum, used in different study

and serum sample would be more convenient for detection and more authentical for

reflecting the status of circulating free radicals. The consistent trends of GSH-Px and

reduced GSH among the observed HIV/HCV groups supported the validity of these

detections in serum samples. In HIV infection, the level of intracellular glutathione in T

cell subsets was decreased and CD4+/CD8+ T cells with higher levels of intracellular

glutathione were selectively lost along with progress of HIV infection. Our study

indicated that both serum oxidant (MDA and GSSG) and antioxidant (GSH-Px and

reduced GSH) parameters were elevated in chronic HIV/HCV-coinfected patients, except

serum zinc. We speculated that, in chronic HIV infection, the frequently destruction of

lymphocytes and red cells may release amounts of MDA, GSH-Px, GSSG and reduced

GSH. This may partially explain why serum antioxidant indicators were not increased in

chronic HCV infection while tremendous increases were found in HIV/HCV coinfection.

However, as an oxidized GSH, serum GSSG is shown to be the best index to reflect the

status of oxidative stress both in HCV and HIV infection, since it is increased distinctly

in chronic HCV-infected, HCV-resolved and HIV/HCV-coinfected patients in our study.

The issue of hepatotoxicity in HIV/HCV-coinfected patients has evolved to a

focus in recent years. The higher morbidity and mortality closely related to HCV

infection have emerged in coinfected patients with highly active antiretroviral therapy

(HAART). We also declared that HCV-related ESLD was a leading cause of mortality in

HIV/HCV-coinfected patients with HAART in comparison with HCV-monoinfected

population (Table 1). However, whether antiretroviral therapy performed a negative or

positive effect on liver hepatitis of coinfected patients was still controversial27. On one

hand, HAART-driven immune reconstitution might retard HIV replication in

lymphocytes resident in liver, which theoretically would contribute to the ameliorative

microenvironment of liver. In consistent with this opinion, some retrospective studies

have reported that slower progression of liver damage would emerge in HAART patients.

On the other hand, the restored immune function might enhance the pre-existing anti-

HCV-specific immune responses in HAART treated HIV/HCV coinfected patients. And

also, HAART-associated drug-induced liver injury (DILI) might worsen HCV-related

hepatitis in a small number of patients, especially patients treated with didanosine,

nevirapine and efavirenz28-32. Our present study clearly showed that HIV coinfection did

increase serum levels of oxidative stress in chronic HCV-infected patients and HCV

resolvers. However, no correlations between serum antioxidants or oxidant parameters

and CD4+T counts were found in HIV-coinfected patients in our study (data not shown),

indicating that higher oxidative stress status in HIV/HCV-coinfected patients might

ascribed to complicated reasons, such as impaired immune system, direct HIV/HCV

cytotoxicity, iron load, HAART application and/or others.

Our data revealed that higher APRI and FIB-4 were presented in HIVpos HCV-

infected patients compared with HIVneg HCV-infected counterparts. Additionally, these

two indicators were strongly consistent since a close correlation was observed (data not

shown) in HCV monoinfection and HIV/HCV coinfection. The ultrasound data also

indicated that aggravated liver parenchyma lesions were presented in HIV-infected

patients than HIV negative individuals, both in chronic HCV carriers and HCV resolvers.

Interestingly, correlations between APRI/FIB-4 and serum GSSG were only found in

HIV-coinfected patients, but not in chronic HCV infection and HCV resolvers. These

data indicated that in HIV/HCV-coinfected patients, the worsening liver fibrosis status

might be HIV-related, and at least partially correlated with the higher levels of serum

oxidant molecules, such as GSSG and MDA. Meanwhile, only higher levels of serum

ALT/AST in HIV/HCV dual infection were consistent with higher levels of serum GSSG

and this phenomenon was not presented in HCV-monoinfected patients. We speculated

that higher concentrations of circulating pro-oxidative components induced by HIV

coinfection should continuously and adversely influence hepatic microenvironment in

HCV-infected patients. Among the possible causes contributed to the aggressive liver

damage initially derived from HCV infection in HIV/HCV coinfected subjects, the

worsen status of oxidative stress induced by HIV was not the sole cause, but should be an

indispensable precipitating factor.

Our study has some limitations. Only a very small number of HIV-monoinfected

patients with negative anti-HCV were found in the same village, as a result there was no

enough sample size to perform convincing statistical analysis and HIV monoinfected

subjects were not included in this study. Since HCV is more efficiently transmitted by

blood-borne routes than HIV, it is easily conceived that most HCV infection, even acute

HCV recovery, occurred prior to HIV infection through contaminated blood. This is why

quite a few HIV positive patients with traces of HCV acute-recovery were found in our

cohort. Actually, near 100% of HIVpos patients in our cohort simultaneously had infected

by HCV (positive anti-HCV at least). In addition, we didn't conduct fibroscan

examination due to limited medical resources at local medical care.

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类别：临床免疫检验906209 外周血 Th17/Treg与手足口病相关性Meta分析

任静静 1），王云凤 1），路璐 1），梁晨 3），石瑛 1，2）#

1）郑州大学第三附属医院检验科 郑州 450052 2）郑州大学检验系 郑州 450052

3）郑州大学第三附属医院儿科 郑州 450052

# 通讯作者：石瑛，研究方向：免疫学检验，E-mail：syybr@126.com

【摘要】目的 研究手足口病（Hand, foot and mouth disease，HFMD）外周血中 Treg/Th17细胞失衡与手足口病患儿机体免疫功能的关系，以期为应用免疫制剂提供依据。方法 检索 EMbase

、Pubmed、Medline、中国知网、万方数据库、维普等建库至 2018年 3月的相关数据，搜集手足口病与 Th17细胞和 Treg细胞相关的病例—对照研究，根据纳入排除标准筛选文章，并采用Newcastle- Ottawa Scale（NOS）文献质量评价工具评估纳入研究。采用 Stata13.0软件进行Meta分析。结果 共纳入 6个病例—对照研究，581例患者。Meta分析结果显示：手足口病患儿和对照儿童外周血 Th17细胞百分率、Treg细胞百分率及 Treg/Th17比例的效应合并值分别为[SMD=1.52，95%CI（0.96，2.07），P=0.000]、[SMD=-1.05，95%CI（-1.42，-

0.68），P=0.000]、[SMD=-1.38，95%CI（-2.11，-0.66），P=0.000]。这一差异同样存在于亚组分析中：对照组 vs. 手足口病普通型的 Th17细胞百分率、Treg细胞百分率及 Treg/Th17细胞比例 SMD（95%CI）分别为[SMD=1.59，95%CI（0.86，2.31），P=0.000]、[SMD=-

0.72，95%CI（-1.16，-0.29），P=0.001]、[SMD=-1.10，95%CI（-1.75，-0.45），P=0.001]；对照组 vs. 手足口病重症型的 Th17细胞百分率、Treg细胞百分率及 Treg/Th17细胞比例SMD（95%CI）分别为[SMD=3.11，95%CI（1.21，5.01），P=0.001]、[SMD=-

1.48，95%CI（-2.32，-0.65），P=0.001]、[SMD=-1.36，95%CI（-2.49，-0.23），P=0.018]；手足口病普通型 vs. 重症型的 Th17细胞百分率 SMD（95%CI）为

[SMD=1.48，95%CI（0.37，2.59），P=0.009]，差异均有统计学意义。结论 HFMD患儿存在细胞免疫功能的调节障碍，外周血 Treg/Th17细胞比例失调，Th17细胞百分率增高以及 Treg

细胞百分率降低，进而为临床应用免疫制剂治疗提供循证学依据。【关键词】手足口病；HFMD；Th17细胞；Treg细胞；Meta

Correlation between peripheral blood Th17/Treg cells and hand, foot and mouth disease: a meta

analysis

Ren Jing-Jing, Wang Yun-Feng, Lu Lu ,Shi Ying

Department of Clinical Laboratory Medicine, The Third Affiliated Hospital of Zhengzhou University,

Henan Maternal and Child Health Hospital, Zhengzhou 450052, China (Shi Ying, Ren Jingjing, Wang

Yunfeng, Lu Lu); Department of Immunology, Zhengzhou University, Zhengzhou 450052,China (Shi

Ying)

【 Abstract 】 Objective To study the relationship between Treg / Th17 cell imbalance and the

immune function of children with hand, foot and mouth disease (HFMD). Methods The data of

EMbase, Pubmed, Medline, CNKI, Wanfang Database, Weipu Database and other libraries were

collected in May 2018. The quality evaluation of the quality assessment tool for Scale Newcastle-

Ottawa Scale (NOS) was used to evaluate the methodological quality of the study. Meta analysis was

performed using Stata13.0 software. Results A total of 6 case-control studies were included in the

analysis of 581 subjects. Meta-analysis showed that the percentages of Th17 cells, the percentage of

Treg cells and the Treg/Th17 ratio in HFMD children and the control were [SMD = 1.52, 95% CI

(0.96, 2.07), P = 0.000], [SMD = -1.05, 95% CI (-1.42, -0.68), P = 0.000], [SMD = -1.38, 95% CI (-

2.11, -0.66), P = 0.000]. This difference was also found in the subgroup analysis: Respectively, these

indicators in the common HFMD and the control were[SMD=1.59, 95%CI(0.86,2.31),P=0.000],

[SMD=-0.72,95%CI(-1.16,-0.29),P=0.001],[SMD=-1.10,95%CI (-1.75,-0.45),P=0.001]; Respectively,

these indicators in the severe HFMD and the control were [SMD=3.11,95%CI(1.21,5.01),P=0.001],

[SMD=-1.48, 95%CI(-2.32,-0.65),P=0.001],[SMD =-1.36,95%CI(-2.49,-0.23),P=0.018]; the

percentages of Th17 cells in the common HFMD and the severe HFMD were[SMD=1.48,95%CI

(0.37,2.59), P=0.009], the differences were statistically significant. Conclusions There is a disorder in

the regulation of cellular immune function, the proportion of Treg / Th17 cells in peripheral blood, the

percentage of Th17 cells and the percentage of Treg cells in HFMD patients, then provide the

evidence for clinical application of immunotherapy.

Key words hand, foot and mouth disease; HFMD; Th17 cells; Treg cells; Meta

手足口病（Hand, foot and mouth disease，HFMD）是由多种肠道病毒引起的急性传染病，临床表现一般为手、足、口腔等部位皮疹、疱疹，伴或不伴发热，轻症者一般具自愈能力，表现为自限性手足口病或疱疹性咽峡炎，为普通型手足口病；但重症型手足口病伴发神经系统感染，病情进展至危重可导致患儿死亡[1,2]。目前手足口病的发病机制未明[3]，尚无特异治疗手段[4]，临床一般采用抗病毒对症支持疗法，有文献支持[5-8]加入调节免疫力的药物往往可明显改善病情，所以HFMD患儿体内免疫功能状态的研究成为近年研究的热点。近年研究[6-11]已证实，HFMD患者的 Th17细胞百分率升高，另一些研究[5-9]显示，HFMD患者的 Treg细胞百分率降低，但目前变化幅度不一，需要进一步研究。该研究拟用循证医学研究方法，通过整合文献中病例—对照研究来增大样本量和效应值，旨在为合理应用免疫制剂治疗HFMD提供循证学依据。1 材料与方法1.1 资料 文献检索中英文电子文献数据库[中国知网、万方数字化期刊全文、维普、Embase、PubMed和Web of science数据库]。中文检索词主题词：手足口病、HFMD、Th17细胞、Treg细胞、对照；英文检索主题词：Hand, foot and mouth

disease、Th17 cell、Treg cell、Control group。时间为自建库至 2018年 3月。1.2 文献纳入和排除标准 纳入标准：①病例—对照研究。②手足口病诊断符合诊断标准或有实验室依据。③文献发表的语言为中文或英文。④原始数据提供外周血Th17细胞、Treg细胞的百分率，且表示为均数±标准差的形式。⑤设置有健康对照

组。排除标准：①摘要、综述、讲座、会议以及述评类的文章。②无健康对照组及病例组仅含重症病例。③缺乏完整数据，数据表述形式为中位数或定性描述。④原始数据表示为 Th17、Treg细胞与 CD4+比例。⑤重复发表的数据，采纳数据详实者。1.3 原始文献的筛选及质量评价 两名评价人员独立按纳入与排除标准筛选文献，纳入病例对照研究的质量评价采用渥太华纽卡斯尔量表（NOS）[12]，量表包括 3大块共 8个条目，具体包括研究人群选择、可比性、暴露评价或结果评价。NOS 对文献质量的评价采用了星级系统的半量化原则，满分为 9 颗星。1.4 资料提取 采集纳入文献的有关信息并制定表格，包括：观察指标的均数和标准差、病例组和对照组的基本资料。1.5 统计学方法 采用 Stata13.0软件进行Meta分析。对各研究的效应进行合并，计算出加权均数差（standardized mean difference）SMD值和 95%置信区间（confidence interval）CI。采用 X2检验检测纳入研究间异质性，结合 I2定量判断异质性大小，在排除明显临床异质性后，可根据异质性检验结果选用固定效应模型或者随机效应模型。采用敏感性分析检验结果稳健性。若纳入文献＞9篇，采用漏斗图分析和 Egger's检验检测发表偏倚。2 结果2.1 文献检索结果 最初检索出与此次Meta 分析相关的文献共 62篇，根据纳入标准和排除标准，最终纳入 6个病例—对照研究，共 623个病例。文献筛选流程及结果见图 1。

通过数据库检索获得相应相关文献（n=62）：CNKI（n=14）、VIP（n=12）、WanFang Data（n=11）、PubMed（n=2）、EMbase（n=7）、Spring（n=16）

通过其他资源补充获得相关文献（n=0）

剔重后获得文献（n=26）

阅读文题和摘要初筛（n=15）

排除会议摘要（n=1）排除无关文献（n=2）

阅读全文复筛（n=12）

纳入定性分析的文献（n=6）

排除不符合纳入标准文献（n=6）

纳入定量合成（Meta分析）的文献（n=6）

图 1.文献筛选流程及结果2.2 纳入研究的基本特征与偏倚风险评价 纳入研究的基本特征与偏倚风险评价结果见表 1。

表 1 纳入研究的基本特征及偏倚风险评价表纳入研究 对照组 手足口病组 Th17细胞百分率（%） Treg细胞百分率（%） Treg/Th17（%） NOS 评分

n(男/女） age（年） n（男/女）

age（年） 对照组 手足口病组 对照组 手足口病组 对照组 手足口病组

阎纳新 2015[6] 30（17/13） 1.52±0.73 15（8/7） 1.96±0.67

0.27±0.22 0.47±0.41 6.55±3.78 2.41±2.17 32.78±20.79 7.07±6.77 7

杨莉 2014[7] 30（17/13） 1.52±0.73 75（41/34

）1.91±0.85

0.27±0.22 0.58±0.74 6.55±3.78 3.21±5.02 33.68±21.40 15.63±23.03 7

刘晶晶 2015[8] 90 267 2.8±0.4 4.79±1.08 6.7±1.3 5.24±1.84 6

冯爱东 2016[9] 31（18/13） 1.50±0.72 35（21/14

）1.87±0.86

0.18±0.08 1.54±0.92 6.6±3.64 2.1±1.64 43.68±30.19 2.76±4.97 7

卢思敏 2016[10] 65（32/33）

3.3±0.5 162（85/7

7）3.3±0.5

0.2±0.06 0.74±0.34 8

盛放 2014[11] 32（18/14） 3.1±1.7 69（39/30

）3.1±2.0

0.19±0.04 0.72±0.34 8

2.3 Meta分析结果2.3.1 外周血 Th17和 Treg细胞百分率与手足口病发病的关系 综合研究结果经Meta

分析显示，手足口病患者外周血 Th17 细胞百分率与对照组相比升高

[SMD=1.52，95%CI（0.96，2.07），P=0.000，见图 2]；而手足口病患者外周血Treg 细 胞 百 分 率 与 对 照 组 相 比 降 低 [SMD=-1.05 ， 95%CI （ -1.42 ， -

0.68），P=0.000，见图 2]；手足口病患者外周血 Treg/Th17细胞比例与对照组相比降低[SMD=-1.38，95%CI（-2.11，-0.66），P=0.000，见图 2]。在各组研究之间，异质性均很明显，故采用随机效应模型进行统计。2.3.2 亚组分析 外周血 Th17、Treg细胞百分率及 Treg/Th17比例在手足口病的普通病例组与对照组、重症病例组与对照组、普通病例组与重症病例组进行比较，在各组研究之间，异质性均很明显，故采用随机效应模型进行统计，见图 3、图 4、图5。Th17细胞百分率的Mata分析结果显示，对照组 vs. 普通病例组、对照组 vs. 重症病例组、普通病例组 vs. 重症病例组合并效应值 SMD（ 95%CI）分别为[SMD=1.59 ， 95%CI （ 0.86 ， 2.31 ） ， P=0.000] 、[SMD=3.11 ， 95%CI （ 1.21 ， 5.01 ） ， P=0.001] 、[SMD=1.48，95%CI（0.37，2.59），P=0.009]，即 Th17细胞百分率：重症病例组＞普通病例组＞对照组，两组差异有统计学意义（P＜0.05）。 Treg细胞百分率的Mata分析结果显示，对照组 vs. 普通病例组、对照组 vs. 重症病例组、普通病例组 vs. 重症病例组合并效应值 SMD（95%CI）分别为 [SMD=-

0.72， 95%CI（ -1.16， -0.29）， P=0.001]、 [SMD=-1.48， 95%CI（ -2.32， -

0.65），P=0.001]、[SMD=-0.73，95%CI（-1.84，0.37），P=0.194]，即 Treg细胞

百分率：对照组＞普通病例组，对照组＞重症病例组，两组差异有统计学意义（P

＜0.05）；但普通病例组与重症病例组比较差异无统计学意义（P＞0.05）。Treg/Th17比例的Mata分析结果显示，对照组 vs. 普通病例组、对照组 vs. 重症病例组 合 并 效 应 值 SMD （ 95%CI ） 分 别 为 [SMD=-1.10 ， 95%CI （ -1.75 ， -

0.45），P=0.001]、[SMD=-1.36，95%CI（-2.49，-0.23），P=0.018]，即 Treg/Th17

比例：对照组＞普通病例组，对照组＞重症病例组，两组差异有统计学意义（P＜0.05）。2.3.3 敏感性分析 在手足口病与对照组比较的基础上，敏感性分析结果显示 Th17

和 Treg细胞百分率的预测效应值均未超过上下限，提示定性描述结果稳健，见图 6

和图 7。

NOTE: Weights are from random effects analysis

.

.

.

Th17 for HFMD

(2016)冯爱东

(2015)刘晶晶

(2016)卢思敏

(2014)盛放

(2015)阎纳新

(2014)杨莉

Subtotal (I-squared = 90.0%, p = 0.000)

Treg for HFMD

(2016)冯爱东

(2015)刘晶晶

(2015)阎纳新

(2014)杨莉

Subtotal (I-squared = 63.3%, p = 0.043)

Treg/Th17 for HFMD

(2015)阎纳新

(2014)杨莉

(2016)冯爱东

Subtotal (I-squared = 79.7%, p = 0.007)

ID

Study

2.02 (1.42, 2.62)

2.08 (1.80, 2.37)

1.87 (1.53, 2.20)

1.87 (1.38, 2.37)

0.68 (0.04, 1.31)

0.49 (0.06, 0.91)

1.52 (0.96, 2.07)

-1.63 (-2.19, -1.07)

-0.85 (-1.10, -0.60)

-1.24 (-1.91, -0.57)

-0.71 (-1.14, -0.28)

-1.05 (-1.42, -0.68)

-1.47 (-2.16, -0.78)

-0.80 (-1.24, -0.36)

-1.95 (-2.54, -1.36)

-1.38 (-2.11, -0.66)

SMD (95% CI)

15.53

18.07

17.73

16.48

15.16

17.03

100.00

21.26

34.92

17.48

26.35

100.00

30.46

36.55

32.98

100.00

Weight

%

2.02 (1.42, 2.62)

2.08 (1.80, 2.37)

1.87 (1.53, 2.20)

1.87 (1.38, 2.37)

0.68 (0.04, 1.31)

0.49 (0.06, 0.91)

1.52 (0.96, 2.07)

-1.63 (-2.19, -1.07)

-0.85 (-1.10, -0.60)

-1.24 (-1.91, -0.57)

-0.71 (-1.14, -0.28)

-1.05 (-1.42, -0.68)

-1.47 (-2.16, -0.78)

-0.80 (-1.24, -0.36)

-1.95 (-2.54, -1.36)

-1.38 (-2.11, -0.66)

SMD (95% CI)

15.53

18.07

17.73

16.48

15.16

17.03

100.00

21.26

34.92

17.48

26.35

100.00

30.46

36.55

32.98

100.00

Weight

%

0-2.62 0 2.62

Th17 Treg and Treg/Th17 for HFMD、

control HFMD

图 2.手足口病和对照组外周血 Th17、Treg细胞百分率及 Treg/Th17比例的比较

NOTE: Weights are from random effects analysis

.

.

.

Th17

(2015)阎纳新

(2014)杨莉

(2015)刘晶晶

(2016)卢恩敏

(2014)盛放

Subtotal (I-squared = 91.7%, p = 0.000)

Treg

(2015)阎纳新

(2014)杨莉

(2015)刘晶晶

Subtotal (I-squared = 60.4%, p = 0.080)

Treg/Th17

(2015)阎纳新

(2014)杨莉

Subtotal (I-squared = 54.1%, p = 0.140)

ID

Study

0.68 (0.04, 1.31)

0.46 (-0.07, 1.00)

2.13 (1.81, 2.44)

2.27 (1.88, 2.67)

2.29 (1.70, 2.88)

1.59 (0.86, 2.31)

-1.24 (-1.91, -0.57)

-0.74 (-1.28, -0.20)

-0.45 (-0.71, -0.19)

-0.72 (-1.16, -0.29)

-1.47 (-2.16, -0.78)

-0.80 (-1.35, -0.26)

-1.10 (-1.75, -0.45)

SMD (95% CI)

18.92

19.78

21.23

20.75

19.31

100.00

23.91

29.80

46.29

100.00

44.65

55.35

100.00

Weight

%

0-2.88 0 2.88

Th17 Treg and Treg/Th17 for common HFMD、

control common HFMD

图 3. 手足口病普通病例组和对照组外周血 Th17、Treg细胞百分率及 Treg/Th17比例的比较

NOTE: Weights are from random effects analysis

.

.

.

Th17

(2014)杨莉

(2016)冯爱东

(2015)刘晶晶

(2016)卢恩敏

(2014)盛放

Subtotal (I-squared = 98.0%, p = 0.000)

Treg

(2014)杨莉

(2016)冯爱东

(2015)刘晶晶

Subtotal (I-squared = 90.2%, p = 0.000)

Treg/Th17

(2014)杨莉

(2016)冯爱东

Subtotal (I-squared = 88.8%, p = 0.003)

ID

Study

0.58 (0.12, 1.04)

2.02 (1.42, 2.62)

6.80 (6.06, 7.54)

2.88 (2.37, 3.38)

3.35 (2.54, 4.16)

3.11 (1.21, 5.01)

-0.73 (-1.20, -0.26)

-1.63 (-2.19, -1.07)

-2.08 (-2.43, -1.72)

-1.48 (-2.32, -0.65)

-0.80 (-1.27, -0.33)

-1.95 (-2.54, -1.36)

-1.36 (-2.49, -0.23)

SMD (95% CI)

20.21

20.05

19.84

20.17

19.73

100.00

33.28

31.86

34.86

100.00

51.23

48.77

100.00

Weight

%

0-7.54 0 7.54

Th17 Treg and Treg/Th17 for severe HFMD、

control severe HFMD

图 4. 手足口病重症病例组和对照组外周血 Th17、Treg细胞百分率及 Treg/Th17比例的比

NOTE: Weights are from random effects analysis

.

.

Th17

(2014)杨莉

(2015)刘晶晶

(2016)卢恩敏

(2014)盛放

Subtotal (I-squared = 96.5%, p = 0.000)

Treg

(2014)杨莉

(2015)刘晶晶

Subtotal (I-squared = 93.9%, p = 0.000)

ID

Study

0.22 (-0.26, 0.69)

2.85 (2.51, 3.20)

1.27 (0.93, 1.62)

1.55 (1.00, 2.10)

1.48 (0.37, 2.59)

-0.15 (-0.63, 0.33)

-1.28 (-1.55, -1.01)

-0.73 (-1.84, 0.37)

SMD (95% CI)

24.82

25.37

25.37

24.44

100.00

48.44

51.56

100.00

Weight

%

0-3.2 0 3.2

Th17 and Treg for common HFMD vs. severe HFMD

common HFMD severe HFMD

图 5. 手足口病普通病例组和重症病例组外周血 Th17及 Treg细胞百分率的比较

0.72 1.52 0.96 2.07 2.24

(2016)冯爱东

(2015)刘晶晶

(2016)卢思敏

(2014)盛放

(2015)阎纳新

(2014)杨莉

Lower CI Limit Estimate Upper CI Limit Meta-analysis estimates, given named study is omitted

-1.75 -1.05 -1.42 -0.68 -0.57

(2016)冯爱东

(2015)刘晶晶

(2015)阎纳新

(2014)杨莉

Lower CI Limit Estimate Upper CI Limit Meta-analysis estimates, given named study is omitted

图 6.外周血 Th17细胞百分率的敏感性分析 图 7.外周血 Treg细胞百分率的敏感性分析3.讨论 目前研究显示，手足口病与 T淋巴细胞亚群水平密切相关，其中 Th17细胞和Treg细胞是 CD4+T细胞的两个亚群，两者功能互逆，在各类自身免疫性及炎症性疾病中起重要作用[13,14]，是最近研究热点。

手足口病是肠道病毒感染的急性疾病，目前临床治疗一般为抗病毒感染、提高患儿免疫功能及对症处理三个原则。EV71感染患儿易致重症型手足口病，大量资料显示重症型手足口病患儿的诊断需及时，在评估机体免疫状态后需尽快用药 [15-21]。相继有文献表明，检测手足口病患儿 Th17细胞及 Treg细胞百分率水平可以评估患儿机体免疫状态，进而为合理应用免疫调控制剂治疗手足口病提供理论依据。但相对于对照组，手足口病患儿的 Th17细胞及 Treg细胞百分率变化的幅度有很大不同，且各个研究样本量、年龄及性别比例不同，故本研究试图进一步扩大样本研究结果，通过合并效应量以明确外周血 Th17细胞及 Treg细胞百分率变化是否可作为手足口病应用免疫调控制剂治疗的实验室依据。此次Meta分析结果显示，手足口病患儿与对照组儿童的 Th17细胞百分率、Treg

细胞百分率及 Treg/Th17细胞比例的效应合并值分别为 1.52（0.96，2.07）、 -

1.05（-1.42，-0.68）、-1.38（-2.11，-0.66），提示HFMD患儿外周血 Th17细胞百分率高于对照组，Treg细胞百分率及 Treg/Th17细胞比例低于对照组，但各研究间异质性很大。进一步亚组分析结果提示，手足口病普通病例组、重症病例组的Th17细胞百分率高于对照组，Treg细胞百分率及 Treg/Th17比例水平均低于对照组，这可能与手足口病患儿的临床表现轻重不一所导致的患儿机体免疫状态不一致有关。敏感性分析前后合并效应量仍都具有统计学意义且森林图结果方向均未改变，说明结果稳健。但是此次Meta分析也存在一定局限性，符合纳入标准的只有中文

文献，可能存在语言偏倚；此外，纳入研究进行亚组分析时仍有亚组存在异质性，推测是由于原始数据中未控制病例组与对照组的年龄、性别比例混杂因素。综上所述，本研究利用Meta分析方法探讨了手足口病患儿外周血 Th17细胞及Treg细胞百分率变化水平。通过病例—对照研究进行效应值合并，提示手足口病患儿机体免疫细胞 Treg/Th17细胞比例失调，Th17细胞百分率升高及 Treg细胞百分率降低，手足口病重症型患儿的免疫细胞变化更为显著。提示手足口病患儿存在细胞免疫功能的调节障碍，进而为临床应用免疫调控制剂治疗提供循证学依据。

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类别：临床免疫检验916406 缺氧相关的microRNAs在子痫前期发病机制中作用的研究进展

李爱萍，何燕霞2

郑州大学第二附属医院，河南郑州，450000

摘要 子痫前期(preeclampsia，PE)是妊娠期特有的一种疾病，也是导致孕产妇和新生儿发病及死亡的主要原因之一。迄今为止，PE的发病机理尚未清楚。微小RNA(microRNA，miRNA)是一类内源性短链非编码小分子 RNA，由 18-23个核苷酸组成。越来越多的研究表明，在缺氧条件下，一些 miRNA在 PE患者胎盘中会存在差异性表达，影响胎盘滋养细胞的侵袭和胎盘血管形成，导致胎盘缺血缺氧。本文将对与缺氧相关的 miRNAs(miR-210、miR-155、miR-18b)在子痫前期发病机制中作用的研究进展进行阐述。关键词 缺氧；miRNA；子痫前期；发病机制Research advance of hypoxia-related miRNAs in the pathogenesis of preeclampsia

Li Ai-ping, He Yanxia.

Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, Henan

Province, China

Abstract Preeclampsia(PE) is a pregnancy-specific syndrome and is one of the main

causes leading to maternal and neonatal morbidity and mortality. So far,the pathogenesis

of PE has not clarified.MicroRNAs(miRNA),is a class of noncoding small RNA

molecules, approximately including 18-23 nucleotides.More and more evidence have

been shown that many miRNAs are expressed differentially in the human placenta with

preeclampsia under hypoxia,impacting invasion of trophoblast cells and placental

angiogensis and causing severe placental ischaemia and hypoxia.Here,we’ll discuss the

progress about the function of hypoxia-related miRNAs(miR-210, miR-155, miR-18b)in

the pathogenesis of PE.

Keywords hypoxia; miRNA; preeclampsia; pathogenesis

2作者单位： 450000 河南 郑州，郑州大学第二附属医院检验科通信作者：何燕霞(1961-)，女，主任技师，硕士生导师 Email : heyanxia1961@163.com

子痫前期(preeclampsia，PE)是正常血压妇女在妊娠 20周以后出现高血压、蛋白尿等临床表现的一种特发性高血压综合征，可引起严重的母胎并发症，也是导致孕产妇和围生儿死亡的主要原因之一[1]。目前，欧美国家子痫前期发病率较低，大约为 2%～5%，而发展中国家子痫前期却更常见，发病率约为 10%，亚洲和非洲子痫前期占孕产妇死亡率的 9%[2]。每年全世界 1000万个孕产妇中由于妊娠期高血压，特别是子痫前期，造成 76000例孕产妇死亡和 500000个胎儿或新生儿的死亡[3]。美国心脏病学会也曾将子痫前期纳入心血管疾病发生的危险因素中，并建议了解有子痫前期病史的患者并改善其生活方式 [4-5]。但子痫前期的发病机制至今仍不清楚，而且临床上尚缺乏有效的治疗手段，很多情况下不得不提前终止妊娠，造成妊娠失败，许多流行病学研究 [6-9]也证明妊娠期子痫前期的发生可能对后代产生长远的不良后果。1 子痫前期的发病机制

PE的发病机制可涉及胎盘缺血缺氧、炎症反应、与 PE相关的信号通路的改变、滋养细胞浸润异常、母胎界面免疫异常、胎盘的局部凝血和抗凝血机制失衡以及microRNA的表达谱异常等多种因素[10-11]。许多研究都认为该病发生的中心环节是胎盘的形成，因为当胎盘娩出后，相关的临床症状会逐渐消失。其中胎盘滋养细胞侵袭异常导致子宫螺旋动脉重塑障碍，胎盘浅着床，进而导致胎盘缺血缺氧，这是PE发生的病理基础。PE发病机制的“两阶段”学说是一个被普遍接受的病因学理论，该学说正是针对胎盘缺血缺氧过程的[12]。妊娠早期，胎盘需氧量较大，因此，胎盘在形成过程中处于一个相对低氧的水平。PE发生时，滋养细胞侵袭异常，部分平滑肌细胞不能被滋养细胞替代，导致胎盘血管形成受到抑制，子宫螺旋动脉重塑失败，胎盘血流灌注不足，而造成胎盘缺血缺氧，胎盘浅着床。而后，胎盘缺血缺氧及坏死的滋养细胞产生了细胞毒性物质，随血液循环到达全身，损伤血管内皮细胞，造成了全身血管弥漫性的损伤，引起全身小动脉的痉挛，发生一系列病理变化，导致胎儿发育迟缓或胎儿窘迫。因此，胎盘的缺血缺氧可能是引起 PE的关键

因素[13]。2 缺氧相关miRNA与 PE相关性的发现 微小 RNA(microRNA， miRNA)是一组长约 18-23个核苷酸的内源性非编码小RNA 分 子 ， 通 过 与 信 使 RNA(mRNA) 的 3' 端 非 编 码 区 (3'-untranslated

region，3'UTR）进行完全或不完全互补配对，起转录后调节基因表达的作用 [14-

15]。miRNA广泛存在于真核生物中，其生物学特性包括进化的高度保守性、表达的时间空间特异性等。miRNA在生长发育、细胞增殖、分化、凋亡等各种生物学过程中发挥重要作用。 自 1993年，Lee等人在秀丽隐杆线虫中发现第一个miRNA至今[16]，miRNA在炎症和肿瘤方面的研究已取得了长足的进展。在肿瘤细胞的研究中，检测到了在低氧条件的诱导下表达上调的 miRNAs[17]。许多研究也均证明了在缺氧条件下某些miRNA的表达发生上调或下调，这些表达情况受低氧调控的 miRNAs称为低氧反应小 RNA或缺氧诱导小 RNA。最近，一些研究 PE发生时，滋养细胞侵袭异常，胎盘血管形成受到抑制，子宫螺旋动脉重塑失败，胎盘血流灌注不足，而造成胎盘缺血缺氧，此时会出现多种 miRNA的表达[2]，参与细胞的增殖、凋亡、分化等生物过程。最近几年的研究也说明了胎盘缺血缺氧与 miRNAs差异性表达之间的关系已经开始引起人们的关注。3 缺氧相关miRNA在 PE 发病中的作用3.1 miR-210 miR-210是在目前所有受低氧处理细胞中上调最为明显的 miRNA，被公认为低氧的标志性miRNAs[18]。miR-210由位于 11p15.5染色体AK123483的基因编码[19],并通过其靶基因调节细胞的代谢、增殖、凋亡等多个生物学过程。在缺氧条件下，miR-210会对滋养细胞的增殖起抑制作用，并且可以较长时间抑制线粒体的新陈代谢，减弱 DNA损伤修复的能力，在子痫前期患者胎盘功能紊乱中起重要作用[20-21]。有研究显示，在缺氧的情况下，miR-210在人胎盘组织中的含量急剧升高,且随病情的严重程度而持续上升[22-23]，提示其对滋养细胞的迁移和侵袭能力可能

具有重要作用，在 PE的发病机制中胎盘的缺血缺氧也被认为是一个关键环节。Luo等[24]等通过生物信息学预测发现 1 型血小板反应蛋白 7A 域(TSHD7A)可能是miR-210的下游靶基因，经检测，在 PE胎盘组织中 TSHD7A的表达水平显著下调，且与miR-210呈负相关关系；进一步研究证实在缺氧情况下，HTR8/SVneo细胞中miR-210的表达水平可显著增加并抑制 TSHD7A的表达。说明了胎盘滋养细胞在缺氧时，miR-210可通过调节靶基因的功能参与 PE的发生。Zhang[25]等研究发现miR-210可通过抑制靶基因 EphrinA3和HOXA9 抑制滋养细胞的侵袭和迁移，影响子宫螺旋动脉的重铸。不仅在胎盘中 miR-210有这种差异性表达，Anton[26]等还发现，在 PE临床症状出现前几个月孕妇血清中 miR-210的含量就可升高，因此，miR-210也是一个具有预测和诊断价值的生物学标志物。这些研究均提示miR-210

表达水平的变化与胎盘缺血缺氧密切相关并参与子痫前期的发生发展。3.2 miR-155 miR-155位于人类 21号染色体上 BIC基因的第三个外显子内[27]，它是一种多功能microRNA，与肿瘤、妊娠期高血压疾病的发生发展密切相关，可能参与多种肿瘤细胞、滋养层细胞的增殖、迁移等过程。目前很多研究都有发现 miR-

155在 PE胎盘中有显著的差异性表达，尤其在重度 PE胎盘中，miR-155呈高表达。Zhang[28]等采用 qRT-PCR方法发现子痫前期患者胎盘中 miR-155显著升高，采用western blots方法检测出 CYR61蛋白的显著下降，证明了 miR-155与 CYR61之间呈负相关关系。富含半胱蛋白 61(CYR61)，也即 CNN1，是 CNN蛋白家属中的一种，是一种重要的血管形成因子，也是一种缺氧敏感性基因，并且在细胞的增殖、迁移、分化和粘附、新生血管形成等多种细胞的生理过程中发挥重要作用，是胎盘缺氧和氧化应激的一个经典通路。因此，胎盘中过度表达的 miR-155可能通过下调CYR61的水平，引起滋养层细胞的血管生成以及炎症调节紊乱，参与 PE的发生、发展。Dai[29]等认为 PE患者滋养细胞中miR-155的过表达可下调 cyclin D的水平，导致滋养细胞凋亡过度、侵袭不足，进而促进子痫前期的发展。cyclin D是重要的细胞周期蛋白，也是影响细胞增殖、凋亡功能的决定性因素之一。因此，研究

cyclin D与miR-155之间的关系，为进一步解释子痫前期形成过程的分子机制奠定了基础。3.3 miR-18b miR-18b 是一段大小约为 20-24nt 的非编码单链 RNA 分子，位于13q31.3染色体上，Dar[30]等的最新研究发现增加黑色素瘤细胞中miR-18b的表达量可提高 p53的表达水平，同时抑制了黑色素瘤细胞的增殖和侵袭能力、诱导其凋亡，由此看出 miR-18b在影响细胞功能中有重要作用。以往也有研究采用高通量的miRNA表达谱芯片技术比较 PE胎盘组织和正常胎盘组织中差异表达的 miRNA，结果发现miR-18b在子痫前期胎盘中呈病理性低表达，以此推测 miR-18b在子痫前期的发生、发展中发挥作用[31]。同样地，Kumar[32]等也发现妊娠早期当胎盘处于相对缺氧时，miR-18b表达增加，可能会促进滋养细胞的增殖；而在妊娠 9-10周后，胎盘缺氧状况加重，miR-18b表达减少，干扰滋养细胞正常的分化过程，促进 PE

的发生。Dolt[33]等发现miR-18b可调控缺氧诱导因子(HIF-1α)的表达。在子痫前期的发生发展过程中，胎盘组织存在不同程度的缺血缺氧，而 HIF-1α参与了低氧相关反应基因的调节，由此推测 miR-18b可能通过靶向调控 HIF-1α基因在子痫前期的发病机制中发挥关键作用。还有研究发现 ERα可能是 miR-18b的靶基因之一[34]，雌激素受体 α(ERα)在人滋养细胞中有表达，且在细胞分化阶段表达增加。雌激素受体效应是通过雌激素受体 α(ERα)与雌激素结合发挥其生物学效应，雌激素在胎盘滋养细胞的侵袭和凋亡等过程中发挥关键作用。而雌激素受体的异常表达可能会导致异常妊娠，如子痫前期的发生。但目前相关研究仍较少，也无靶基因验证的相关研究，因此 ERα是否为 miR-18b的靶基因仍不确定，无法判定两者是否有直接的的靶向调控关系。4 展望子痫前期的发病机制涉及多种复杂的因素，如胎盘的缺血缺氧，而且其发病机

制至今仍不十分清楚，这也为临床的治疗带来了困扰。在对子痫前期胎盘缺血缺氧机制的研究过程中，一些研究发现了参与子痫前期发病的 miRNAs，如 miR-

210、miR-155、miR-18b等，并且对这些在胎盘组织中差异表达的 miRNAs进行了靶标预测，目前发现的相关的靶基因有 HIF-1α、CYR61、VEGF等。因而对缺氧相关miRNA的靶基因进行进一步的探索研究，发现这些靶基因在胎盘滋养细胞侵袭及胎盘血管形成过程中发挥的作用，并进一步在体外细胞水平或进行动物实验构建子痫前期的模型，验证这些缺氧相关的miRNAs是如何在子痫前期的发生发展过程中起作用的，从而有可能设计开发针对这些 miRNAs的靶向药物，干预相关靶基因的表达，缓解子痫前期发病过程中的缺血缺氧状况，改善子痫前期的预防和治疗水平。参考文献[1] Ghulmiyyah L,Sibai B.Maternal mortality from preeclampsia/eclampsia[J].Semin Perinatol,

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类别：临床免疫检验842430 不同甲胎蛋白分层的初治原发性肝癌的临床特点分析

姜艳红　陈光意　盛家和　许青霞＃郑州大学附属肿瘤医院检验科 河南郑州 450008

摘要 　 目的：分析不同甲胎蛋白（AFP）分层的初治原发性肝癌（PLC）患者临床特征的差异，并分析有差异的临床特征与AFP分层的相关性。方法：回顾性分析 2012年 1月至 2015年 12月河南省肿瘤医院肝胆外科 452例确诊初发 PLC住院病人的临床病历资料，采用 Chi-square test比较不同AFP分层的临床特征的差异，并采用 Spearman秩相关分析有差异的临床特征与不同AFP分层的相关性。结果：对于不同的AFP分层，年龄、HBV、肿瘤大小、脉管瘤栓、肝被膜受侵、周围侵犯、病理组织学分型和 TNM分期不同分组间差异均有统计学意义（χ2值和 P值分别为：11.057，0.011；23.659，0.000；32.549，0.000；19.800，0.000；8.839，0.032；7.848，0.049；37.678，0.000；16.558，0.011；P均＜0.05），但性别、糖蛋白抗原 199（CA199）、肿瘤数、Child-Pugh分级和巴塞罗那临床肝癌（BCLC）分期不同分组间差异均无统计学意义（χ2值和 P值分别为：0.567，0.904；1.206，0.752；10.449，0.107；0.453，0.929；5.464，0.486；P均＞0.05）。HBV、肿瘤大小、脉管瘤栓、肝被膜受侵和 TNM分期不同分组与AFP分层存在等级正相关关系（r值和 P值分别为：0.200，0.000；0.190，0.000；0.190，0.000；0.125，0.008；0.104，0.027；P均＜0.05），但年龄和病理组织学分型不同分组与AFP分层存在等级负相关关系（r值和 P值分别为：－0.104，0.026；－0.234，0.000；P均＜0.05）。结论：对于不同的AFP分层，初治PLC患者的年龄、HBV、肿瘤大小、脉管瘤栓、肝被膜受侵、周围侵犯、病理组织学分型和 TNM分期的构成是不同的，而且年龄、HBV、肿瘤大小、脉管瘤栓、肝被膜受侵、病理组织学分型和 TNM分期的不同分组与不同AFP分层存在等级相关关系。关键词：原发性肝癌，甲胎蛋白，乙型肝炎病毒，临床特征，相关性Analysis of clinical features of de novo primary liver cancer with different stratification of alpha fetal proteinJiang Yanhong, Chen Guangyi, Sheng Jiahe, Xu Qingxia＃ Clinical Laboratory, The Tumor Hospital of Zhengzhou University, Zhengzhou, Henan 450008, ChinaAbstract Objective: To investigate the clinical features of de novo primary liver cancer(PLC) with different stratification of alpha fetal protein(AFP), and analyze the correlations between the different clinical features and the stratification of AFP. Methods: Clinical data of 452 confirmed new PLC patients in the tumor hospital of Henan between January 2012 to December 2016 was retrospectively reviewed and analyzed by chi-square test, and the correlations between the different clinical features and the stratification of AFP were analyzed by Spearman rank correlation. Results: Comparing the

stratification of AFP, there were statistically significant differences in age, HBV, tumor size, vascular invasion, liver capsule invasion, perineural invasion, histopathological typing and TNM staging(χ2 value and P value: 11.057, 0.011; 23.659, 0.000；32.549,0.000; 19.800, 0.000; 8.839, 0.032; 7.848, 0.049; 37.678, 0.000; 16.558, 0.011; P<0.05), but there were no statistically significant differences in sex, CA199, tumor number, Child-pugh staging and BCLC staging（χ2 value and P value：0.567, 0.904; 1.206, 0.752; 10.449, 0.107; 0.453, 0.929; 5.464, 0.486; P>0.05）. Analyzed by Spearman rank correlation, there were positive rank correlations between the five differentiated clinical features(HBV, tumor size, vascular invasion, liver capsule invasion and TNM staging) and the stratification of AFP（r value and P value: 0.200, 0.000; 0.190, 0.000; 0.190, 0.000; 0.125, 0.008; 0.104, 0.027; P<0.05）, but negative rank correlations between the two features(age and histopathological typing) and the stratification of AFP（r value and P value: -0.104, 0.026; -0.234, 0.000; P>0.05）. Conclusion: There were different proportions in age, HBV, tumor size, vascular invasion, liver capsule invasion, perineural invasion , histopathological typing and TNM staging among the stratification of AFP, furthermore, there were rank correlations between the seven clinical features(age, HBV, tumor size, vascular invasion, liver capsule invasion,histopathological typing and TNM staging) and the stratification of AFP.Key words：Primary liver cancer(PLC), Alpha fetal protein(AFP), Hepatitis B virus(HBV), Clinical feature, Correlation　　原发性肝癌(Primary liver cancer，PLC)是进展速度快、预后不良的恶性肿瘤之一，居于全球癌症病死率第三位［1］。2008年最新统计结果显示，每年全球新发肝癌人数约为 74.8万，而因肝癌死亡的病人高达 69.6万[2-3]。我国是肝炎大国，也是PLC的高发区，PLC在肿瘤相关死亡中仅次于肺癌，位居第二[4]。甲胎蛋白（alpha fetal protein，AFP）作为临床上应用最为广泛的肝癌标志物，不仅用于肝癌的筛查和诊断，而且对肝癌术后的预后判断、监测复发转移也具有重要价值[5]。本文通过把AFP进行分层，对不同 AFP分层的 PLC临床特点进行回顾性分析，旨在进一步提高对该病的认识，为临床早期诊断提供依据，现将分析结果报道如下。资料和方法一、对象　　收集 2012年 1月至 2015年 12月河南省肿瘤医院肝胆外科所有确诊初发 PLC住院病人 1365例，PLC严格按照《原发性肝癌诊疗规范 2011 版》诊断标准进行诊断，通过查阅病历筛选资料完备的病例，最终有 452例病例符合要求，男 375例（83.0%），女 77例（17.0%），年龄范围 23～81岁，中位年龄 52岁。二、标本采集与处理 　　用促凝采血管采集病人术前空腹静脉血 2ml，离心分离血清备用。三、方法 　　用罗氏电化学发光分析仪 E601检测 HBV相关项目，用 E602检测 AFP和CA199；通过查阅病历收集病人年龄、性别、肿瘤数、肿瘤大小、脉管瘤栓、肝被受侵、周围侵犯和病理学诊断结果。根据 Child-Pugh 分级标准对患者肝功能情况

进行分级，采用巴塞罗那临床肝癌（Barcelona Clinic Liver Cancer,BCLC）分期标准和 TNM分期标准进行分期。四、统计学处理 　　应用 SPSS17.0软件进行统计分析，计数资料的比较采用 Chi-square test（χ2 检验），采用 Spearman秩相关进行二元变量的等级相关分析，检验水准 α＝0.05。结果一、临床病理特征分析

PLC病人血清 APF（ng/ml）按≦20、20～200、200～400和＞400进行分层分组，各组病人数（例）分别为 159、83、28和 182。AFP不同分层男性病人数（例）分别为 130、71、23和 151，女性分别为 29、12、5和 31，差异无统计学意义（P=0.904＞0.05）；PLC病人年龄按≦65岁和＞65岁分组，AFP不同分层≦65岁病人数（例）分别为 138、77、22和 172，＞65岁分别为 21、6、6和 10，差异有统计学意义（P=0.011＜0.05）；PLC病人按有无乙型肝炎分组，AFP不同分层HBV阴性组病人数（例）分别为 42、8、3和 16，HBV阳性组分别为 117、75、25和 166，差异有统计学意义（P=0.000＜0.05）；Ca199血清含量（ng/ml）按≦30和＞30进行分组，AFP不同分层≦30病人数（例）分别为 107、56、16和 119，＞30组分别为 52、27、12和 63，差异无统计学意义（P=0.752＞0.05）；肿瘤数按1 个、 2 个和多个分组， AFP 不同分层 1 个肿瘤组病人数（例）分别为127、67、23和 129，2个分别为 8、9、3和 23，多个分别为 24、7、2和 30，差异无统计学意义（P=0.07＞0.05）；肿瘤直径（cm）按≦5、5～10和＞10分组，AFP 不同分层≦5组病人数（例）分别为 71、47、16和 52，5～10组分别为66、26、9和 75，＞10组分别为 22、10、2和 55，差异有统计学意义（P=0.000＜0.05）；PLC病人按脉管瘤栓有无分组，AFP不同分层无瘤栓组病人数（例）分别为 149、79、25和 146，有瘤栓组分别为 10、4、3和 36，差异有统计学意义（P=0.000＜0.05）；肝被膜按有无受侵分组，AFP 不同分层无受侵组病人数（例）分别为 41、22、7和 26，有受侵组分别为 118、61、21和 156，差异有统计学意义（P=0.032＜0.05）；PLC病人按肝脏周围有无侵犯分组，AFP不同分层无侵犯组病人数（例）分别为 142、79、25和 152，有侵犯组分别为 17、4、3和30，差异有统计学意义（P=0.049＜0.05）；病理学诊断按肝细胞癌、胆管细胞癌和混合型细胞癌分组，AFP不同分层肝细胞癌病人数（例）分别 ,125、77、28和174，胆管细胞癌组分别是 28、2、0和 5，混合型细胞癌组分别是 6、4、0和 3，差异有统计学意义（P=0.000＜0.05）；Child-Pugh分级 A和 B，AFP不同分层 A级组分别为 148、79、26和 170，B 级组分别是 11、4、2和 12，差异无统计学意义（P=0.929＞0.05）；TNM分期Ⅰ、Ⅱ、Ⅲ和Ⅳ，AFP不同分层Ⅰ期组分别为99、59、15和 91，Ⅱ期组分别为 3、3、1和 3，Ⅲ期组分别为 47、20、12和 84，Ⅳ期组分别为 10、1、0和 4，差异有统计学意义（P=0.011＜0.05）；BCLC分期0、A、B 和 C 期，AFP 不同分层 0期组分别为 2、2、0和 0，A期组分别为

99、59、16和 91，B期组分别为 20、10、4和 26，C期组分别为 38、12、8和65，差异无统计学意义（P=0.486＞0.05）（表 1）。表 1　不同AFP分层的 PLC临床特征

临床特征 AFP分层 统计值 P值≦20 20～200 200～400 ＞400

性别（例） 0.567 0.904

男 130 71 23 151

女 29 12 5 31

年龄（例） 11.057 0.011

≦65 138 77 22 172

＞65 21 6 6 10

HBV（例） 23.659 0.000

无 42 8 3 16

有 117 75 25 166

Ca199 1.206 0.752

≦30 107 56 16 119

＞30 52 27 12 63

肿瘤数（例） 10.449 0.1071 127 67 23 129

2 8 9 3 23≧3 24 7 2 30

肿瘤直径（例） 32.549 0.000

≦5 71 47 16 52

5～10 66 26 9 75

＞10 22 10 3 55

脉管瘤栓（例） 19.800 0.000

无 149 79 25 146

有 10 4 3 36

肝被膜受侵（例） 8.839 0.032

无 41 22 7 26

有 118 61 21 156

周围侵犯（例） 7.848 0.049

无 142 79 25 152

有 17 4 3 30

病理学诊断（例） 37.678 0.000

肝细胞癌 125 77 28 174

胆管细胞癌 28 2 0 5

混合型细胞癌 6 4 0 3

Child-Pugh分级（例） 0.453 0.929

A 148 79 26 170B 11 4 2 12

TNM分期（例） 16.558 0.011

Ⅰ 99 59 15 91Ⅱ 3 3 1 3Ⅲ 47 20 12 84Ⅳ 10 1 0 　　4

BCLC分期（例） 5.464 0.4860 2 2 0 0A 99 59 16 91B 20 10 4 26C 38 12 8 65

二、有差异临床特征与AFP分层相关性的分析对 AFP分层中有差异的临床特征与 AFP分层进行 Spearman秩相关二元变量的

等级相关分析。PLC病人年龄按≦65岁和＞65岁分组，分别赋值 1和 2，与 AFP分层有等级负相关性(r＝－0.104，P＝0.026＜0.05)；PLC病人按 HBV阴性和阳性分组，分别赋值 1和 2，与AFP分层有等级正相关性(r＝0.200，P＝0.000＜0.05)；肿瘤直径（cm）按≦5、5～10和＞10分组，分别赋值 1、2和 3，与AFP分层有等级正相关性(r＝0.190，P＝0.000＜0.05)；PLC病人按脉管瘤栓无和有分组，分别赋值 1和 2，与 AFP分层有等级正相关性(r＝0.190，P＝0.000＜0.05)；肝被膜按无和有受侵分组，分别赋值 1和 2，与 AFP分层有等级正相关性(r＝0.125，P＝0.008＜0.05)；PLC病人按肝脏周围无和有侵犯分组，分别赋值 1和 2，与AFP分层无等级相关性(r＝0.086，P＝0.068＞0.05)；病理学诊断按肝细胞癌、胆管细胞癌和混合型细胞癌分级，分别赋值 1、2和 3，与 AFP分层有等级负相关性(r＝－0.234，P＝0.000＜0.05)；TNM分期Ⅰ、Ⅱ、Ⅲ和Ⅳ，分别赋值 1、2、3和 4，与 AFP分层有等级正相关性(r＝0.104，P＝0.027＜0.05)（表 2）。表 2　有差异临床特征与AFP分层相关性的分析

临床特征 AFP分层r P值≦20 20～200 200～400 ＞400

年龄（例） -0.104 0.026≦65 138 77 22 172

＞65 21 6 6 10

HBV（例） 0.200 0.000

无 42 8 3 16

有 117 75 25 166

肿瘤直径（例） 0.190 0.000

≦5 71 47 16 52

5～10 66 26 9 75

＞10 22 10 3 55

脉管瘤栓（例） 0.190 0.000

无 149 79 25 146

有 10 4 3 36

肝被膜受侵（例） 0.125 0.008

无 41 22 7 26

有 118 61 21 156

周围侵犯（例） 0.086 0.068

无 142 79 25 152

有 17 4 3 30

病理学诊断（例） -0.234 0.000

肝细胞癌 125 77 28 174

胆管细胞癌 28 2 0 5

混合型细胞癌 6 4 0 3

TNM分期（例） 0.104 0.027

Ⅰ 99 59 15 91Ⅱ 3 3 1 3Ⅲ 47 20 12 84

Ⅳ 10 1 0 　　4

讨论　　血清甲胎蛋白（alpha fetal protein，AFP）是一种人胚胎发育期分泌的糖蛋白，在正常成人的血清中含量极低，临床上多以 AFP＞20ng/ml为阳性，当肝细胞发生癌变时大约有 60%的患者AFP 可表现为阳性,本组资料 452例 PLC患者中血清AFP 阳性（＞20ng/ml）者 293例（64.8%），接近于国内研究水平 [6]。有学者研究表明，术前 AFP阴性患者的肝癌恶性程度低于阳性者，AFP 阴性患者的术后复发率较低，但生存率明显高于 AFP 阳性组，特别是术前 AFP＞400ng/mL 的 PLC 患者，远期疗效不佳[7]。本文通过把患者血清 AFP含量按≦20、20～200、200～400和＞400进行分层，来分析不同 AFP分层 PLC患者的临床特征，提高对 AFP与 PLC关系的认识，为临床 PLC的早期诊断和治疗提供帮助。 原发性肝癌（primary hepatic carcinoma，PLC）是世界范围内发病率和死亡率均较高的恶性肿瘤之一[8]，主要诱因包括：HBV 和HCV 感染、黄曲霉素、亚硝胺类物质、酒精和肝硬化等，但 HBV 感染和酗酒是诱发肝癌最主要的原因[9]。有全基因组关联性研究结果显示，针对人类基因组不同位点的单核苷酸多态性与慢乙肝患者进展为肝细胞癌 PLC有[10,11]。慢性 HBV感染区域与肝癌高发区域有极大的重合之处，高 HBV感染区域同时也是肝癌的高发区 [12]。本文 PLC患者乙肝阳性 383例，占 84.7%，乙肝阴性 69例，占 15.3%，与以上文献相符，统计学结果显示，对于AFP 的不同分层，乙肝阴性与阳性组之间的差异有统计学意义（P＝0.000＜0.05），且与AFP不同分层有等级正相关性（r＝0.200，P＝0.000＜0.05）。巴西的一项关于 PLC发病率和危险因素的长达 10年的队列研究结果指出，

PLC的发病以男性为主，平均年龄为 57岁左右[13]。本文 PLC患者男性 375例，占83.0%，女性 77例，占 17.0%，中位年龄 52岁，与上述文献报道相符，统计学结果显示，对于 AFP的不同分层，性别差异无统计学意义（P＝0.904＞0.05），但年龄差异有统计学意义（P＝0.011＜0.05），且与 AFP不同分层有等级负相关性（r＝－0.104，P＝0.026＜0.05）。Li等 [14]对我国初发的 588例早发型 HBV相关性PLC进行了危险因素分析，结果发现在相同年龄组内，男性、有 HBV感染家族史、AFP＞200ng/ml是 PLC的危险因素，男性 PLC患者明显多于女性患者可能与性激

素有关，雄激素可通过介导 DNA损伤和氧化应激诱发肝癌，相反雌激素可抑制肝癌的发生[15]。糖蛋白抗原 199是一种低聚糖类抗原，最初是 Denilano 从胰腺癌病人的血清中

发现的，在消化道肿瘤如肝癌、胰腺癌、结肠癌等患者的血清中浓度明显升高，而在正常人血清中含量很低。近年来，有越来越多的研究开始关注 CA199 与肝硬化和肝癌的关系。有研究发现其在 PLC中敏感度为 38%～70%[16]，本文 PLC总例数452例，CA199阳性 154例（敏感度为 34.1%），与上述文献数据接近。也有研究表明 CA199 血清水平与乙肝、丙肝或肝硬化疾病进程密切相关[17]，统计学结果显示，对于 AFP 的不同分层，CA199 阴性与阳性组之间的差异无统计学意义（P=0.752＞0.05）。 有学者研究发现，PLC肿瘤直径越大，病理分级就越高，细胞分化程度就越低，肿瘤恶性度就越高，预后就越差[18]。也有研究表明，肿瘤直径大、数目多和门脉受侵犯是治疗后 PLC 复发的最常见不良因素[19]。本文数据显显示，对于 AFP的不同分层，不同肿瘤数分组间差异无统计学意义（P=0.107＞0.05），但不同肿瘤大小分组间差异有统计学意义（P＝0.011＜0.05），且与 AFP不同分层有等级正相关性（r＝0.190，P＝0.000＜0.05）。

门静脉癌栓（portal vein tumor thrombus，PVTT）是影响 PLC预后极为重要的不良因素之一，是 PLC的严重并发症及转移方式。PVTT在 PLC发生率较高，有文献报道 10%～40%的 HCC患者存在镜下门静脉癌栓 [20-21]。本文 PLC共 452例，其中发现 PVTT的有 53例（11.7%），与上述文献相符，对于 AFP的不同分层，PVTT阴性与阳性组之间的差异有统计学意义（P=0.000＜0.05），且与 AFP不同分层有等级正相关性（r＝0.190，P＝0.000＜0.05）。肿瘤大小与肝被膜是否侵及反映了肿瘤的负荷程度，多项研究结果表明，HCC

侵及肝被膜是肿瘤复发的不良预后因素之一 [22]，本文数据显示对于 AFP的不同分层，肝被膜受侵阴性与阳性组之间差异有统计学意义（P＝0.032＜0.05），且与AFP不同分层有等级正相关性（r＝0.125，P＝0.008＜0.05）。临近肝被膜的癌结节可侵犯临近器官和组织，如胃、膈、结肠、胸腔等，本文数据显示对于 AFP的不同分层，周围组织器官侵犯阴性与阳性组之间差异有统计学意义（P＝0.049＜0.05），但与 AFP不同分层无等级相关性（r＝0.086，P＝0.068＞0.05）。Child-Pugh 肝功能分级法是国际上通用的衡量肝脏储备功能的分级标准，主要应用于病情的估计、治疗效果的评定及手术承受能力的判断等；BCLC分期与治疗策略，比较全面地考虑了肿瘤、肝功能和全身情况，与治疗原则联系起来, 并且具有循证医学高级别证据的支持，目前已在全球范围被广泛采用，本文数据显示对于 AFP的不同分层，Child-Pugh分级之间和 BCLC分期之间的差异均无统计学意义（P分别为 0.929和 0.486，均＞0.05）。　　PLC按病理组织学可分为：肝细胞型肝癌、胆管细胞型肝癌和两者混合型肝癌，在我国，大多数 PLC患者属于肝细胞癌（96.8%） [23]。本文 PLC共 452例，

HCC404例（89.4%），与上述文献接近，统计结果显示，对于 AFP的不同分层，PLC病理组织学分组之间的差异有统计学意义（P＝0.000＜0.05），且与 AFP不同分层有等级负相关性（r＝－0.234，P＝0.000＜0.05）。　　TNM分期主要根据肿瘤的大小、数目、血管侵犯、淋巴结侵犯和有无远处转移而分为Ⅰ-Ⅳ期,由低到高反映了肿瘤的严重程度； 其优点是对肝癌的发展情况做了详细的描述, 最为规范，然而 TNM分期在国际上被认可程度却较低。本文统计结果显示，对于 AFP的不同分层，TNM分期之间的差异有统计学意义（P＝0.011＜0.05），且与 AFP 不同分层有等级正相关性（ r＝0.104，P＝0.027＜0.05）。　　综上所述，对于不同的 AFP分层，PLC患者的年龄、HBV、肿瘤大小、脉管瘤栓、肝被膜受侵、周围侵犯、病理组织学分型和 TNM分期的构成是不同的，而且年龄、HBV、肿瘤大小、脉管瘤栓、肝被膜受侵、病理组织学分型和 TNM分期的不同分组与不同AFP分层存在等级相关关系。参考文献[1]Block TM, Mehta AS, Fimmel CJ, et al. Molecular viral oncology of hepatocellular carcinoma[J]. Oncogene, 2003, 22(33): 5093-5107. DOI:10.1038/sj.onc.1206557.[2]Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002[J].CA Cancer J Clin, 2005, 55(2): 74-108. DOI:10.1002/aic.690451118.[3]Ferlay J, Shin HR, Bray F, et al. Estimates of worldwide burden of cancer in 2008: Globo Can 2008[J]. Int J Cancer, 2010, 127(12): 2893-2917. DOI:10.1002/ijc.25516.[4]Tang ZY. Perspective of clinical oncology from the viewpoint of liver cancer studies[J]. Tumor, 2009, 29(1): 1-4. DOI:10.3781/j.issn.1000-7431.2009.01.001.[5]Nomura F, Ohnishi K, Tanabe Y. Clinical features and prognosis of hepatocellular carcinoma with reference to serum alpha-fetoprotein levels. Analysis of 606 patients[J]. Cancer, 1989, 64(8): 1700-1707.[6]樊庆胜, 王茂强. 原发性肝癌肿瘤标志物对疗效、转移和预后评估的研究进展[J]. 临床医学工程, 2011, 18(1): 152-154. DOI:10.3969/j.issn.1674-4659.2011.01.0152.[7]马文骏. 术前血清AFP水平与肝细胞肝癌生物学特性及预后相关性分析[D]. 浙江大学, 2014.[8]Parkin DM. Global cancer statistics in the year 2000[J]. Lancet Oncol, 2001, 2(9): 533-543.[9]Myers RP, Shaheen AA, Hubbard JN, et al. Characteristics of patients withcirrhosis who are discharged from the hospital against medical advice[J]. ClinGastroenterol Hepatol, 2009, 7(7): 786-792. DOI:10.1016/j.cgh.2009.03.020.[10]Zhang H，Zhai Y Hu Z，et a1 ． Genome-wide association study identifies lp36.22 as a new susceptibility locus for hepatocellular carcinoma in chronic hepatitis B virus carriers[J] ． Nat Genet，2010, 42(9)：755-758. DOI:10.1038/ng.638.[11]Chan KY, Wong CM, Kwan JS, et a1 ． Genome-wide association study of hepatocellular carcinoma in Southern Chinese patients with chronic hepatitis B virus infection. PLoS One, 2011, 6(12):e28798. DOI: 10.1371/journal.pone.0028798[12]Kew MC．Epidemiology of chronic hepatitis B virus infection，hepatocellular carcinoma，and hepatitis B virus-induced hepatocellular carcinoma[J] ． Pathol Biologie， 2010, 58(4)： 273-277. DOI:10.1016/j.patbio.2010.01.005.[13]Paranagua-Vezozzo DC，Ono SK，Alvarado-Mora MV, et a1 ． Epidemiology of HCC in Brazil：incidence and risk factors in a ten-year cohort[J]．Ann Hepatol，2014，13(4)：386-393.

[14]Li Y Zhang Z，Shi J， et a1 ． Risk factors for naturally-occurring early-onset hepatocellular carcinoma in patients with HBV-associated liver cirrhosis in China[J] ． Int J Clin Exp Med，2015，8(1)：1205-1212.[15]Kengvw, Largaespadada, Villanuevaa ． Why men are at higher risk for hepatocellular carcinoma[J]．J Hepatol，2012，57(2)：453-454. DOI:10.1016/j.jhep.2012.03.004.[16]代维，李志清，姜红峰，等. 血清 AFP、CA19-9、CA125、CEA、r-GT联合检测对原发性肝癌的诊断价值［J］．中华中医学杂志，2007，31(2): 143-144．[17]Stem A, Laughlin GA, Bergstrom J, etal. The sex-specific association of serum osteoprotegerin and　receptor activator of nuclear factor kappa B legend with bone mineral density in older adults:the Rancho Bermardo study [J]. Eur J Endocrinol, 2007,156(5): 555-562. DOI:10.1530/EJE-06-0753.[18]沈智勇, 吴名凤, 张亚力, 等. 超声测量肝癌大小与术后多预后因素相关分析[J]. 医学影像学杂志, 2011, 21(12): 1842-1845.[19]Colecchia A, Schiumerimi R, Cucchetti A, et al. Prognostic factors for hepatocellular carcinoma recurrence[J]. world J Gastroenterol, 2014, 28: 20(20): 5935-5950. DOI:10.3748/wjg.v20.i20.5935.[20]Cheung TK, Lai CL, Wong BC, et al. Clinical features, biochemical parameters and virological profiles of patients with hepatocellular carcinoma in Hong Kong[J]. Aliment Pharmacol Ther . 2006, 24(4): 573-583. DOI:10.1111/j.1365-2036.2006.03029.x.[21]Minagawa M, Makuuchi M. Treatment of hepatocellular carcinoma accompanied by portal vein tumor thrombus[J]. World J Gastroenterol. 2006, 12(47): 7561-7567. DOI:10.1116/1.581798.[22]吴凡, 王黎明, 吴健雄, 等. 巴塞罗那分期 0-A期肝细胞肝癌术后复发危险因素分[J]. 中华医学杂志，2015，95(22): 1747-1750.[23]丛文铭, 吴孟超. 肝脏肿瘤 3160 例临床病理研究[J]. 中华病理学杂志, 1997, 26(2): 70-73.

类别：临床免疫检验908960 受体相互作用蛋白 3增强结肠癌细胞系HCT116对顺铂敏感性

代淑阳，滑世轩，许波实*阜外华中心血管病医院检验科，河南省郑州市

摘要目的 研究受体相互作用蛋白 3（RIP3）在结肠癌细胞系 HCT116中的表达以及对顺铂敏感性的影响，并探讨其作用机制。方法 免疫荧光、western blot检测 RIP3在HCT116细胞中的表达；构建 RIP3重组质粒，瞬时转染 RIP3重组质粒并用免疫荧光检测外源表达的 RIP3在细胞内的定位；细胞活性试验和 Annexin V-FITC／PI双染色法检测外源表达 RIP3对凋亡、坏死细胞数目的影响;提取转染后细胞全蛋白并用 western blot检测 RIP3介导的蛋白表达的改变并结合实验室前期研究探讨RIP3介导HCT116细胞对顺铂敏感性的作用机制。结果 RIP3在HCT116中表达较低，外源转染 RIP3重组质粒后，RIP3 主要定位与胞浆；细胞活性试验和 Annexin V-FITC／PI双染色法显示外源转染 RIP3可以增强 HCT116细胞对顺铂的敏感性；western blot显示过表达 RIP3后 DNA损伤修复通路相关蛋白表达降低，提示 RIP3可以通过影响 DNA 损伤修复 通路的活性增强顺铂的细胞毒性作用从而增加HCT116细胞对顺铂的敏感性。结论 HCT116细胞中 RIP3的表达降低，过表达RIP3后可以通过介导DNA损伤修复通路的活性增强HCT116细胞对顺铂的敏感性。关键字： 受体相互作用蛋白 3，HCT116，顺铂，DNA损伤修复结肠癌是一种发病率极高的恶性肿瘤，在全球男性和女性恶性肿瘤发病率中分

别居第 2位和第 3位，肿瘤细胞对化疗耐药性的产生是肿瘤治疗的主要障碍。顺铂是一种常用的化疗药物，可以用于多种实体瘤的治疗。顺铂通过与 DNA结合形成DNA加合物(单加合物，链内或链间连接)，阻碍 DNA的复制与转录并诱导细胞发生死亡（1,2）。之前报道顺铂可以通过调控特定离子通道、转运蛋白和多种质膜酶类的活性(3,4)，诱导活性氧（ROS）的产生(5)以及调控 DNA损伤修复、抑制转录及细胞周期阻滞(6)等途径诱导化疗中肿瘤细胞的死亡。最近越来越多的研究表明细胞对顺铂的敏感性不仅取决于顺铂的毒性效应还取决于细胞对 DNA损伤的反应（7）。DNA损伤修复系统是修复顺铂介导的 DNA加合物的主要细胞修复机制，DNA损伤修复系统中的MutSa 复合体(MSH2 and MSH6异源二聚体)可以识别并结合到顺铂介导的 DNA加合物上， MuLa ( MLH1 and PMS2异源二聚体) 然后被募集并招募其他蛋白，协助损伤位点的修复。MMR与 DNA加合物的结合可以增强细胞毒性效应并活化下游信号通路最终导致细胞凋亡。然而，在MMR缺陷的肿瘤细胞中，由于顺铂引起的 DNA加合物被很少修复有助于肿瘤细胞对化疗药物产生耐受（8）。

RIP3是执行坏死性凋亡的关键分子，在 caspase 抑制剂存在的情况下，RIP3与

RIP1结合形成淀粉样结构并相互磷酸化以及磷酸化下游靶蛋白，如混合谱系激酶结构域样蛋白（mixed lineage kinase domain-like protein，MLKL）(9)并最终导致细胞的程序性坏死的发生。RIP3的表达在多种肿瘤中是下调的，由于启动子的甲基化而造成 RIP3的低表达或者不表达抑制了肿瘤细胞对化疗药物的敏感性（10），并有利于肿瘤细胞的取得生存优势。我们通过实验证明了 RIP3在 HCT116细胞中的表达也是下调的，RIP3的下调是否与 HCT116的化疗耐药性有关，以及 RIP3在HCT116细胞化疗耐药性中的确切机制目前还不清楚。我们通过实验证明了外源表达 RIP3可以增强 HCT116细胞对顺铂的敏感性，而且 western blot实验表明过表达RIP3可以导致 CDC37、JNK、ERK 和 AKT 表达降低，结合实验室前期研究和生物信息学分析这些蛋白可以介导 DNA损伤修复通路的活性，因此我们推测，在DNA损伤修复缺陷的 HCT116细胞中，RIP3可能通过增强 DNA损伤修复通路的活性增强顺铂的细胞毒性作用从而增强 HCT116细胞对顺铂的敏感性，以 RIP3位靶点可能成为结肠癌治疗及预后诊断的新靶点。1. 材料和方法1.1 材料与试剂

HCT116 细胞系购自华大基因，胎牛血清（FBS）购自美国 Gibicol 公 司，0．25％Trypsin—EDTA消化液购自美国 Sigma 公司 DH5α 大肠杆菌感受态细胞购自北京天根生物技术公司，兔抗人 RIP3 抗体购自兔抗人 RIP3（ab72106）购自Abcam（Cambridge，UK）。鼠抗人 CDC37（ cat. No. sc-13129）、鼠抗人 p-JNK（cat. No. sc-6254）购自 Santa cruz biotech. （Santa cruz, CA,USA）；兔抗人 p-ERK（ cat. No. 4370）、兔抗人 p-AKT （ cat. No. 9271）购自 Cellsignaling technology（Boston, MA, USA）。辣根过氧化物酶标记山羊抗兔和抗鼠二抗购自中杉金桥生物技术公司（北京，中国）；1.2 实验方法1.2.1 细胞转染细胞培养至融合度达 85%以上，胰酶消化细胞，培养基重悬并调整细胞浓度

为 1x105/ml，将细胞接种至六孔板中，置细胞培养箱中培养 12h待细胞贴壁。取出六孔板，在无菌操作台内，按照说明书要求将 Lipofectamine 2000与 Opti MEM培养基混合液和质粒与 Opti MEM混合液混合，静止 20min，用 1xPBS洗涤六孔板 3次，每孔加入 700μL Opti MEM和 300μL 质粒混合液置细胞培养箱培养 6h，然后每孔加入 1mL含 20%FBS的 1640细胞培养液，继续转染培养。1.2.2 细胞爬片制备和免疫荧光试验无菌玻片放入六孔板中, 每个玻片表面用 0. 01%的多聚赖氨酸处理 30min, 然后

将食管癌 KYSE510-pCMV6和 KYSE510-PURα细胞悬液 1x105/ml加至六孔板中, 混匀, 至 37℃培养箱培养 24h. 取出六孔板, 将细胞爬片用 4℃预冷的 1x磷酸盐缓冲液(PBS)洗涤 3次, 每次 3min, 2% BSA封闭后加入一抗, 4℃孵育过夜后 PBS洗涤 3

次, 每次 3min, 加入二抗, 室温孵育 1h后 PBS洗涤 3次并用 DAPI封片, 显微镜下观察.1.2.3顺铂药物处理与细胞活性测量取两孔分别转染 RIP3和 PCI-neo24h后的 HCT116细胞，胰酶消化，将细胞悬

液离心后各用 3ml培养基重悬，然后在一个 96 孔板中每孔加入 100μl细胞悬液，培 养 24h 待 细 胞 贴 壁 。 然 后 分 设 对 照 组 （ 0μM 顺 铂 ） 和 不 同 浓 度(2.5、5、10、20、40、80μM)的顺铂药物组，每孔设 4 复孔，培养 2h后，每孔加入 10μCCK-8继续培养 2h，680 型全自动酶标仪(Bio—Rad 公司)450nm波长检测吸光度。以对照组细胞活力为 100％，按公式细胞抑制率=1一(药物组吸光度／对照组吸光度)×100％，GraphPad Prism 5计算各组细胞存活率。1.2.4 统计学方法实验数据采用 SPSS 19.0统计软件分析，数据以 x±s表示，各组间比较采用 t

检验，每组内不同处理方式比较采用方差分析，P＜0.05为差异有统计学意义。2 结果2.1 结肠癌细胞系HCT116中 RIP3的表达细胞爬片免疫荧光显示在野生型 HCT116细胞中，RIP3的表达较低，瞬时转

染 RIP3后，RIP3表达量显著升高，且 RIP3 主要为胞浆定位（图 1）.

图 1.瞬时转染 RIP3重组质粒后 RIP3在HCT116细胞中的表达情况2.2 转染 RIP3后细胞对顺铂的敏感性增加

从象限图分析，细胞主要以凋亡性死亡为主（Q2Q4象限，Q2 代表晚期凋亡、Q4 代表早期凋亡），统计分析细胞死亡率，转染 RIP3后细胞的死亡率增高，提示RIP3 促进了 HCT116细胞的凋亡性细胞死亡（图 2）。细胞活性试验显示过表达RIP3后，HCT116细胞对顺铂的敏感性增加（图 3）.

图 2.Annexin V-FITC／PI双染色法检测外源表达 RIP3后HCT116凋亡情况。图 3.CCK-8细胞活性试验检测HCT116细胞对顺铂的敏感性。

3、western blot检测 RIP3转染后细胞损伤修复通路相关蛋白的改变提取过表达 RIP3重组质粒后的HCT116细胞蛋白，western blot试验检测DNA

损伤修复通路相关蛋白表达情况，试验发现，过表达 RIP3后，DNA损伤修复通路相关蛋白 CDC37、JNK、p-AKT和 p-ERK表达下调（图 4），提示 RIP3可能通过下调 DNA损伤修复通路活性增加HCT116细胞对顺铂的敏感性。

图 4. 过表达RIP3 后，HCT116细胞DNA 损伤修复通路相关蛋白表达情况讨论顺铂是一种常用的化疗药物，可以用于多种实体瘤的治疗。顺铂通过与 DNA

结合形成 DNA加合物(单加合物，链内或链间连接)，阻碍DNA的复制与转录并诱导细胞发生死亡（1,2）。细胞对顺铂的敏感性不仅取决于顺铂的毒性效应还取决于细胞对 DNA损伤的反应（7）。之前的研究显示，顺铂可以通过调控特定离子通道、转运蛋白和多种质膜酶类的活性(3,4)；诱导活性氧（ROS）的产生以及调控DNA损伤修复、抑制转录、细胞周期阻滞(Siddik Z.et al,2003)等途径诱导化疗中肿瘤细胞的死亡。Wang X等人发现 ERK信号通路的活化是顺铂介导的细胞凋亡所必需的（ 11） ,Dent P 等人报道 c-Jun N 端激酶（ JNK）、应激活化蛋白激酶（SAPK）和 p38丝裂原活化蛋白激酶（MAPK）在顺铂诱导的肿瘤细胞的凋亡中

被活化（12）.此外在人上皮细胞中，顺铂可以诱导 Fas/FasL介导的细胞凋亡并可以通过调控 Bcl-2 家族蛋白的表达调控线粒体的损伤（13）。研究报道顺铂可以通过增强 GM3的表达并介导线粒体通路参与的氧化应激损伤介导 HCT116细胞的凋亡，过表达GM3可以增强HCT116对顺铂的敏感性（14）。

RIP3是程序性细胞坏死通路中的关键角色，caspase被抑制的细胞受到刺激时可以引起细胞程序性坏死而非凋亡（15)。死亡受体家族如 TNFR1、Fas、死亡受体3（Death receptor3,DR3）、DR4、DR5和肿瘤坏死因子相关凋亡诱导配体（TNF-related apoptosis-inducing ligand receptor,TRAIL）等与其相应配体结合活化后可以引起其胞内结构域发生构象变化并募集一系列蛋白并形成复合体I，复合体I中的RIP1 泛素化可以募集转化生长因子 β 激活激酶 1（Transforming growth factor-βactivated kinase 1, TAK1 ） 、 TAK 结 合 蛋 白 2 （ TAK1-binding protein 2, TAB2)、TAB3并活化NF-κB，促进炎症反应并抑制细胞死亡。当 RIP1去泛素化时，转而与 RIP3、TRADD、Fas死亡机构域相关蛋白（Fas-associated protein via a death domain, FADD）及 caspase-8结合形成复合体II。如果 caspase-8具有正常活性，则caspase-8会裂解 RIP1和 RIP3并引起细胞凋亡；如果 caspase-8被抑制，则 RIP1和RIP3会进一步形成淀粉样结构并相互磷酸化并磷酸化下游一些靶蛋白，其中最终要的靶蛋白是混合谱系激酶结构 域样蛋白（mixed lineage kinase domain-like protein，MLKL）(9)并最终导致细胞的程序性坏死的发生。

RIP3的表达在多种肿瘤中是下调的，由于启动子的甲基化而造成 RIP3的低表达或者不表达抑制了肿瘤细胞对化疗药物的敏感性（10），RIP3是肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）介导的细胞凋亡和细胞坏死之间的分子开关，在凋亡受到抑制时，RIP3可以通过调节能量代谢而介导细胞坏死。因此肿瘤下调RIP3的表达有利于肿瘤细胞的取得生存优势。本实验发现在DNA损伤修复缺陷的HCT116细胞中，通过过表达 RIP3重组质

粒恢复 RIP3的表达可以使 HCT116对顺铂的敏感性显著增加，western blot检测发现 RIP3可能通过降低 DNA损伤修复通路的活性增加顺铂的细胞毒性作用从而增强 HCT116细胞对顺铂的敏感性，因此以 RIP3位靶点可能成为结肠癌治疗及预后诊断的新靶点。参考文献1. Jamieson ER, Lippard SJ (1999) Structure, Recognition, and Processing of Cisplatin–DNA Adducts. Chem Rev 99: 2467–2498.2. Jordan P, Carmo-Fonseca M. Molecular mechanisms involved in cisplatin cytotoxicity. Cell Mol Life Sci 2000;57:1229–35.3. Aggarwal SK, Niroomand–Rad I (1983) Effect of cisplatin on the plasma membrane phosphatase activities in ascites sarcoma-180 cells: a cytochemical study. J Histochem Cytochem 31: 307–317.4. Grunicke H, Hofmann J (1992) Cytotoxic and cytostatic effects of antitumor agents induced at the plasma membrane level. Pharmacol Ther 55: 1–30.5. Masuda H, Tanaka T, Takahama U (1994) Cisplatin generates superoxide anion by interaction with DNA in a cell-free system. Biochem Biophys Res Commun 203: 1175–1180.

6. Siddik ZH (2003) Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 22: 7265–7279.7. Wang D, Lippard SJ. Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov 2005;4:307–20.8. Vaisman A, Varchenko M, Umar A, Kunkel TA, Risinger JI, Barrett JC, et al. The role of hMLH1, hMSH3, and hMSH6 defects in cisplatin and oxaliplatin resistance: Correlation with replicative bypass of platinum-DNA adducts. Cancer Res 1998;58: 3579–85.9. Zhao J, Jitkaew S, Cai Z, et al. Mixed lineage kinase domain- like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proc Natl Acad Sci USA 2012; 109:5322-5327.10. Koo GB, Morgan MJ, Lee DG, Kim WJ, Yoon JH, Koo JS, Kim SI, Kim SJ, Son MK, Hong SS, Levy JM, Pollyea DA, Jordan CT, Yan P, Frankhouser D, Nicolet D, Maharry K, Marcucci G, Choi KS, Cho H, Thorburn A, Kim YS. Methylation-dependent loss of RIP3 expression in cancer represses programmed necrosis in response to chemotherapeutics. Cell Res. 2015 Jun;25(6):707-25.11. Wang X, Martindale JL, Holbrook NJ (2000) Requirement for ERK activation in cisplatin-induced apoptosis. J Biol Chem 275: 39435–39443.12. Dent P, Grant S (2001) Pharmacologic interruption of the mitogen–activated extracellular–regulated kinase/mitogen–activated protein kinase signal transduction pathway: potential role in promoting cytotoxic drug action. Clin Cancer Res 7: 775–783.13. Razzaque MS, Koji T, Kumatori A, Taguchi T (1999) Cisplatin–induced apoptosis in human proximal tubular epithelial cells is associated with the activation of the Fas/Fas ligand system. Histochem Cell Biol 111: 359–365.14. Chung TW, Choi HJ, Kim SJ, Kwak CH, Song KH, Jin UH, Chang YC, Chang HW, Lee YC, Ha KT, Kim CH. The ganglioside GM3 is associated with cisplatin-induced apoptosis in human colon cancer cells. PLoS One. 2014 May 14;9(5)15. Kaiser WJ, Upton JW, Long AB, Livingston-Rosanoff D,Daley-Bauer LP, Hakem R, Caspary T, Mocarski ES. RIP3 mediates the embryonic lethality of caspase-8-deficient mice. Nature, 2011, 471(7338): 368–372.

类别：临床免疫检验871655 绝经后子宫肌瘤患者 IGF-I、ER及 PR水平表达及临床意义研究

殷卫兵 余晨晨 严鸣光（河南省商丘市第一人民医院 检验科 476000）

【摘要】目的：观察绝经后子宫肌瘤患者 IGF-I、ER及 PR水平表达，并分析其临床意义。方法：选取 2015年 1月-2016年 6月我院收治 42例绝经后女性因子宫肌瘤不萎缩反增长行子宫肌瘤切除术患者作为实验组，同期选择 42例子宫脱垂行子宫切除术患者为对照组，比较两组患者血清中雌二醇、孕酮水平表达以及组织标本中 IGF-I、ER、PR阳性率，并分析这些指标与子宫肌瘤发病之间的关系。结果：实验组患者血清的雌二醇、孕酮表达水平明显高于对照组（P<0.05）；实验组的IGF-I、ER、PR阳性率明显高于对照组（P<0.05）。结论：对于绝经后子宫肌瘤患者而言，病变组织内 ER、PR高阳性表达是促进患者子宫肌瘤生长的直接原因；病变组织中 IGF-I的高水平表达同时也是促进子宫肌瘤生长的一个关键因素，IGF-I

的高水平表达直接导致雌二醇、孕酮的非正常表达，进一步进发一系列的连锁反应，促进绝经后子宫肌瘤生长。【关键词】绝经后；子宫肌瘤；生长因子；激素【中图分类号】R737.33 【文献标识码】B

The expression of IGF-I,ER and PR in uterine fibroids in postmenopausal patients

and clinical value

Yin weibing   Yu chenchen    Yan mingguang   

(Department of Clinical Laboratory ,The first people’s hospitol of ShangQiu,

ShangQiu ,Henan  476000 ,China )

Abstract: Objective To observe the expression of IGF-I,ER and PR in uterine fibroids in

postmenopausal patients and its clinical value. Methods 42 uterine fibroids cases in

postmenopausal women, who underwent hysteromyomectomy as observation group were

enrolled in our hospital from 2015.01 to 2016.06, and 42 patients with hysterectomy for

uterine prolapse were selected as control group. To compare the expression rates of E2, P

and IGF-I in two groups, and analyzed its relationship with the uterine fibroids. Results

The serum E2 and P in observation group was higher than that in control group, the

difference was statistically significant (P<0.05). The expression rates of ER, PR and IGF-

I in observation group were significantly higher than in control group (P<0.05).

Conclusion The high positive expression ER, PR in tumor tissue might directly relate to

overgrowth of fibrous tissue in postmenopausal fibroids. High levels of IGF-I might

promote the abnormal expression of E2 and PD, trigger a chain reaction and result in a

overgrowth of connective tissue and development of myoma.

Key words: Post-menopause ; uterine fibroids; hormone; growth factor

引言： 在妇科，子宫肌瘤是一种常见疾病，这一疾病是女性生殖系统中的一种良性肿瘤，是由于患者子宫平滑肌细胞异常增生而导致，好发人群为 30-50岁女性，其中以 40-50岁发病率最高，占 51.2%-60.9%[1]。同时，这一疾病也是女性患者切除子宫的主要原因之一。根据相关资料显示，这一疾病的确切发病因素并未明了，现阶段的研究结论为子宫肌瘤是一种雌激素依赖性肿瘤，雌激素、孕激素均可促进子宫肌瘤细胞进行有丝分裂，并导致瘤体增大；性激素对于子宫肌瘤细胞的有丝分裂也具有一定的促进作用，这一作用主要依赖局部来源的多肽类生长因子 ( EGF、 IGF

等) 通过自分泌 /旁分泌形式介导的。临床研究发现，随着患者年龄的增加，子宫肌瘤逐渐萎缩，但是有部分患者在绝经之后，子宫肌瘤不但不萎缩，反而会逐渐增大，最终只能采取手术治疗。根据相关研究报道显示，与生育期子宫肌瘤患者相比，绝经后子宫肌瘤患者的恶变发生率增高，对绝经后子宫肌瘤患者进行研究发现，子宫肌瘤的发生、发展与雌孕激素以及相应的生长因素有密切关系。为进一步分析绝经后子宫肌瘤患者 IGF-I、ER及 PR水平表达及临床意义，选取我院收治 42例绝经后女性因子宫肌瘤不萎缩反增长行子宫肌瘤切除术患者与 42例子宫脱垂行子宫切除术患者作为研究对象，现报告如下。一 资料与方法1.1 一般资料

选取 2015年 1月-2016年 6月我院收治 42例绝经后女性因子宫肌瘤不萎缩反增长行子宫肌瘤切除术患者作为实验组，同期选择 42例子宫脱垂行子宫切除术患者为对照组。其中实验组年龄 45-65岁，绝经时间为（2.45±1.98）年，子宫肌瘤确诊时间为（2.12±1.58）年。对照组年龄 40-60岁。经统计学分析，除了年龄之外，观察组与对照组的一般资料无统计学意义（P>0.05），本次研究具有可比性。1.2 纳入标准[2-3]

（1）符合子宫肌瘤的诊断标准，均为多发性肿瘤，直径均大于 4cm；（2）患者在术前 3个月内未进行激素类药物治疗；（3）患者经病理学检验，子宫内膜为绝经后的子宫内膜组织；（4）符合子宫切除术的手术指征；（5）患者自愿参与本次研究并签署相关知情同意书。1.3 排除标准[4-5]

（1）合并糖尿病、甲状腺等疾病患者；（2）卵巢肿瘤、宫颈癌等患者；（3）意识障碍的患者。（4）合并严重基础性疾病的患者。1.4 方法1.4.1 实验试剂与仪器 本次用来检测的试剂均来自于福州迈新公司；本次实验所用的仪器主要有石蜡包埋切片机、显微镜、干燥箱、染色机、免疫组化油笔等，均来自于本院实验室。1.4.2操作方法 首先，进行子宫切除术时，采取患者 4ml 静脉血，并采用酶联免疫吸附法，进行E2、P水平的检测；其次，将术后切除的子宫肌瘤中心组织进行石蜡连续切片的制作，采用免疫组化测定 IGF-I、 EＲ 及 PＲ 的表达水平。1.4.3 结果判定标准[6-7]

IGF-I细胞浆出现棕黄色颗粒，为阳性；EＲ及 PＲ细胞核出现棕黄色颗粒，为阳性。在高倍镜下，对每张切片进行随机 10个视野的观察，阳性细胞染色深度如下：细胞未见染色记为 0；细胞轻度染色记为 1；细胞中度染色记为 2；细胞深度染色记为 3。阳性细胞百分率标准如下：＜5%的细胞着色记为 0；5%~细胞着色记为 1；25%~细胞着色记为 2；50~细胞着色记为 3；≥75%的细胞着色记为 4。

最后根据染色强度计分和阳性细胞百分比计分之和所得总分进行结果判定。阴性（-）为 0～1分；弱阳性（+）为 2分；阳性（++）为 3～4分；强阳性（+++）为5～7分。以上所有结果的判定均采用统一标准、双盲法。1.5 血清 E2、P的正常诊断范围[8-9]

正常生育期女性：E2 为 20～528pg /mL；P为 0～25.03ng /mL。绝经期女性：E2 为 40～100pmol /L；P为 0 ~3.2nmol /L。

1.6 统计学处理 应用 SPSS18.0统计学系统进行分析，P＜0.05 代表差异具统计学意义。二 结果2.1两组血清 E2、P表达水平比较实验组患者血清的 E2、P表达水平明显高于对照组（P<0.05），其详细内容见

表 1。表 1两组血清 E2、P表达水平比较[(n)%]

组别 E2（pg /mL） P（nmol /L）实验组（n=42） 128.23±38.24 7.19±0.25

对照组（n=42） 30.43±14.27 1.35±0.82

X² 12.938 35.231

P ＜0.05 ＜0.05

2.2两组 EＲ、PＲ、IGF-I 的阳性情况比较两组患者子宫肌瘤标准的阳性细胞计数统计结果内容见表 2，根据阳性统计结

果显示，实验组的 IGF-I、ER、PR阳性率明显高于对照组（P<0.05），其详细内容见表 3。

表 2两组 EＲ、PＲ、IGF-I 的阳性统计结果比较[(n)%]

组别 ER PR IGF-I

- + ++ +++ - + ++ +++ - + ++ +++

实验组 3 8 19 12 4 7 20 13 1 14 18 9

对照组 15 15 8 4 12 13 11 6 11 15 9 7

表 3两组 EＲ、PＲ、IGF-I 的阳性率比较[(n)%]

组别 ER阳性率 PR阳性率 IGF-I 阳性率实验组（n=42） 39（92.86） 38（90.48） 41（97.62）对照组（n=42） 27（64.29） 30（71.43） 31（73.81）X² 10.127 7.238 8.238

P ＜0.05 ＜0.05 ＜0.05

三 讨论 长期研究以来，E2一直被认为是女性发生子宫肌瘤的主要病因，同时，也被

认为是子宫肌瘤发生与发展的促进剂[10]。在这一研究领域，较多的研究学者达成一项共识，即无论是在临床研究，还是在实验研究中，E2与女性与子宫肌瘤的发生、病情发展具有一定的必然联系。随着子宫肌瘤病因学的积极开展，越来越多的研究

报道显示[11-12]，P在女性子宫肌瘤发病过程中与 E2发生着同样的生理作用，这一结局在组织学、生物化学以及临床临床研究等方面均得到验证。现阶段子宫肌瘤的确切发病因素以及发病机制并不明确，其可能的发病机制[13-14]包括：①： 女性体内的激素，尤其是雌孕激素以及相应的受体在机体内异常水平表达；②患者体内某些生长因子的促进作用导致子宫肌瘤的发生与发展；③原癌基因的促癌作用被启动及抑癌基因的作用受到抑制，或是这两者之间的平衡被打破；④子宫组织细胞凋亡活动受到抑制，导致相应细胞的堆积，进而促进子宫肌瘤的病情的发展。虽然这一可能发病机制进一步显示出患者体内的雌孕激素对于子宫肌瘤的发生与发展具有刺激作用，但是，并没有直接证据证明这一点。近年来的研究报告除了说明雌孕激素在子宫肌瘤中的重要作用，在女性机体中

还存在一些因子以及受体通过影响雌孕激素的方法对瘤体的生长起到一定的促进作用。例如，胰岛素样生长因子- ( IGF-I) Ⅰ 就引起了人们的关注和研究。根据实验证明，这一生长因子能够有效调控细胞的增值情况，对于子宫组织平滑肌细胞的有丝分裂活动具有明显的促进作用，间接促进子宫肌瘤的发生与发展。此外，还有研究表明[15]，IGF-I在子宫肌层、肌瘤组织中含量较多，其性激素的分泌对其有一定的促进作用。本次研究结果显示，在 E2、P水平表达方面，实验组明显高于对照组，这说明

E2、P与子宫肌瘤的发病与密切关系，可能在这一疾病发病过程中具有重要的促进作用，同时，绝经后子宫肌瘤组织中 ER、PR阳性率也明显高于对照组，这进一步说明，在绝经子宫肌瘤患者中，ER、PR对于促进子宫肌瘤的生长具有关键作用。由于子宫肌瘤组织中 E2、P以及相应受体高水平表达，这导致子宫平滑肌上的ER、PR大量分泌，进而促进有丝分裂活动的进行，促进子宫肌瘤的增长。此外，绝经后患者 IGF-I阳性率明显高于对照组，过高的 IGF-I水平表达直接促进性激素分泌，也直接导致子宫肌瘤的增长。因此，绝经期控制 IGF-1 的水平可以预防性激素与受体不正常表达，或许可以在一定程度上控制子宫肌瘤的发生与发展。

综上所述，对于绝经后子宫肌瘤患者肉眼，病变组织内 ER、PR高阳性表达是促进患者子宫肌瘤生长的直接原因；病变组织中 IGF-I的高水平表达同时也是促进子宫肌瘤生长的一个关键因素，IGF-I的高水平表达直接导致 E2、PD的非正常表达，进一步进发一系列的连锁反应，促进绝经后子宫肌瘤生长。参考文献[1]李元成,崔志丹,沈伶. 子宫肌瘤患者外周血胰岛素样生长因子水平的变化及其与子宫肌瘤组织中雌激素受体和孕激素受体的关系研究 [J]. 中国全科医学,2014,15（12）:1728-1730+1738. [2]翟一阳,林琼林. 子宫肌瘤患者外周血胰岛素样生长因子-Ⅰ和Ⅱ水平的变化及与子宫肌瘤组织雌激素受体的关系[J]. 广东医学,2015,22（11）:3516-3518. [3]单思群,章卉琴,谢慧君,施鑫锋. 子宫肌瘤患者外周血 IGF-Ⅰ和 IGF-Ⅱ的表达及其与 ER、PR的相关性分析[J]. 南通大学学报(医学版),2016,03（16）:227-229. [4]陈梦珠,彭丹红. 绝经激素治疗对围绝经期及绝经后子宫肌瘤影响的研究进展[J]. 现代医学,2016,06（23）:882-887. [5]孙华萍. 胰岛素样生长因子-Ⅰ及雌激素受体在绝经后子宫肌瘤中的表达[J]. 中国医药指南,2013,12（16）:167-168. [6]龚宇 ,艾战秀 ,黎锐勤 ,罗虹,徐晓 . 子宫肌瘤患者 IGF-Ⅰ和 IGF-Ⅱ水平的变化与ER、PR的关系研究[J]. 中国医药科学,2016,16（18）:95-97+165. [7]李亮,宋成文,杨赛花,王晶. IGF-Ⅰ、IGF-Ⅱ、ER及 PR在子宫组织的表达及肌瘤复发的意义[J]. 中国妇幼保健,2015,17（16）:2874-2876. [8]李弦,吴晓玲,周美. 子宫肌瘤患者 IGF-Ⅰ和 IGF-Ⅱ水平变化与 ER、PR的关系研究[J]. 河北医药,2015,19（19）:2986-2987. [9]欧阳俊,黄利红. 子宫肌瘤患者外周血胰岛素样生长因子水平与激素受体表达的相关性[J]. 中国妇幼保健,2015,30（02）:5155-5157. [10]蔡云朗,沈扬,任慕兰,赵维英,姚青,何杰,李嘉,翟卫中. 米非司酮对子宫肌瘤组织中雌、孕激素受体及表皮生长因子受体的影响[J]. 现代医学,2007,04（21）:299-303. [11]樊艺,韩克. Ki67 ER及 PR在乳腺癌患者服用他莫昔芬后子宫内膜的表达及其性激素水平的变化[J]. 中国妇幼保健,2012,25（12）:3986-3989. [12]吕华,夏恩兰,成九梅,彭雪冰. 促性腺激素释放激素激动剂对子宫肌瘤及子宫肌层组织中雌、孕激素受体和碱性成纤维生长因子表达的影响 [J]. 中华妇产科杂志,2006,02（11）:135-136. [13]Cui Xiaojiang,Zhang Ping,Deng Wanleng,Oesterreich Steffi,Lu Yiling,Mills Gordon B,Lee Adrian V. Insulin-like growth factor-I inhibits progesterone receptor expression in breast cancer cells via the phosphatidylinositol 3-kinase/Akt/mammalian target of

rapamycin pathway: progesterone receptor as a potential indicator of growth factor activity in breast cancer.[J]. Molecular Endocrinology,2003,174（21）:. [14]Milewicz T,Kolodziejczyk J,Krzysiek J,Basta A,Sztefko K,Kurek S,Stachura J,Gregoraszczuk E L. Cyproterone, norethindrone, medroxyprogesterone and levonorgestrel are less potent local human growth hormone and insulin-like growth factor I secretion stimulators than progesterone in human breast cancer explants expressing the estrogen receptor.[J]. Gynecological Endocrinology,2002,164（16）:. [15]van Lier Elize,Meikle Ana,Eriksson Håkan,Sahlin Lena. Insulin-like growth factor-I (IGF-I) and thioredoxin are differentially expressed along the reproductive tract of the ewe during the oestrous cycle and after ovariectomy[J]. Acta Veterinaria Scandinavica,2006,481（21）:.

类别：临床免疫检验906202 血清游离的轻链比值用于判断冒烟型多发性骨髓瘤预后的价值

谢林森 肖华 黄永杰 梁永钢 史华瑞郑州大学附属郑州中心医院检验科(450007)

冒烟型多发性骨髓瘤是否需早期治疗还有争议，因为令人无法接受的治疗的毒性和没有一个好的指标判断冒烟型多发性骨髓瘤在短期内会发展成为多发性骨髓瘤。文献报道冒烟型多发性骨髓瘤恶性转换的标志物包括浆细胞异常、骨髓中浆细胞比例、m蛋白浓度和血清中游离轻链比率等，血清中游离轻链比率在意义未明的单克隆丙种球蛋白血症、多发性骨髓瘤和浆细胞白血病中增高的范围较宽，本研究的目的就是探讨高血清游离轻链比值作为冒烟型多发性骨髓瘤恶性转变高风险的生物标志物临界值。一、对象与方法对象：回顾分析了 2008年到 2017年郑州大学附属郑州中心医院及郑州市中心

医院医疗集团中 28 家二级以上医院诊断的冒烟型多发性骨髓瘤患者 117例，年龄平均 63岁，男：女 64:53.

方法：血清游离轻链的检测均在郑州市临床检验中心进行，试剂为西门子公司提供（freelite；The binding Site,Birmingham,UK）,设备为西门子公司的 BNⅡ特种蛋白分析仪，k/λ参考区间为 0.26—1.65.

统计学：使用 SPSS21.0进行 ROC曲线和 Chi-square 检验二、结果1. 117例冒烟型多发性骨髓瘤平均随诊时间为 33个月，有 22例患者随访少于

18个月，这 22例中 14例（65%）在随诊期间没有疾病进展，没有发展成多发性骨髓瘤，只有一例轻链比值大于 100，为 317（k9.82mg/l,λ3120mg/l）。117例患者免疫球蛋白类型、游离轻链浓度、m蛋白浓度和骨髓浆细胞比例结果见表一

表一 117例患者免疫球蛋白类型、游离轻链浓度、m蛋白浓度和骨髓浆细胞比例结果特征 所有病人 FLC比值<100 FLC比值≧100 P

血清蛋白M

平均浓度（g/L）

25 25 27 0.76

<30 75（68%） 61（65%） 10（56%） 0.22

≧30 38（32%） 34（35%） 8（4%） 0.22

免疫球蛋白类型IgG 85（73%） 70（74%） 11（65%） 0.09

IgA 23（20%） 19（20%） 3（18%） 0.84

IgM 1（1%） 0（0%） 0（0%） 0.46

IgD 1（1%） 1（1%） 1（1%） 0.59

双克隆 3（2%） 3（3%） 0（0%） 0.12

轻链 4（3%） 2（2%） 3（16%） 〈0.001

骨髓浆细胞比例平均 20 20 30 〈0.001

10-60% 111（95%） 112（96%） 102（88%） 0.002

>60% 6（5%） 5（4%） 15（12%） 0.002

2、冒烟型多发性骨髓瘤血清游离轻链比值≥100发展为多发性骨髓瘤的比率为91%，明显高于血清游离轻链比值﹤100的 44%，其中一年、两年和三年发展为多发性骨髓瘤的比率也明显。

表二 血清游离轻链比值与冒烟型多发性骨髓瘤预后指标 所有病人 游离轻链

〈100游离轻链≧100 RR（95%可信区间）

发展MM（%） 51 44 91 2.04（1.8-2.2）一年内（%） 15 14 40 1.5（1.3-1.8）2年内（%） 32 25 68 2.6（1.8-3.6）3年内（%） 43 36 80 4.4（2.7-7.4）器官损害比率骨损害（%） 40 37 40 1.0（0.84-1.3）

贫血（%） 30 34 21 0.81（0.71-0.94）肾功能损害（%）

17 15 25 1.2（1.0-1.1）高钙血症（%） 5 6 6 1.0（0.95-1.1）

三、讨论无症状骨髓瘤（冒烟型骨髓瘤）的诊断标准血清单克隆 M蛋白≥30g/L和/或骨

髓单克隆浆细胞比例≥10%，无相关器官及组织的损害（无终末器官损害，包括溶骨改变）对于无症状MM建议仅随访，在出现 CRAB以后进行治疗，这是因为既往的治疗模式下，治疗并未带来更多的生存获益，但随着新药的不断出现，例如来那度胺、帕马度胺等，使得早期治疗成为可能，已有临床研究显示早期应用这些药物可以带来总体生存的获益。Mateos等的研究表明，对于出现下列情况之一者，患者多在 2年内进展为MM，属于高危 SMM。高危 SMM的定义如下：1）浆细胞>10%以及免疫球蛋白数值达到以下水平（ IgG>30g/L， IgA>20g/L，轻链>1g/

24h）；2）如果浆细胞>10%或者 Ig数值达到以上水平，意即以上条件仅达到 1 条，则需要骨髓中浆细胞群中 95%为克隆性浆细胞或未受累轻链下降至少 25%以上。临床试验结果证实在这些高危患者中使用 RD（来那度胺联合地塞米松）治疗患者的肿瘤进展时间以及总体生存均可获益，在随访 40个月之后，使用来那度胺联合地塞米松治疗组 3年的总体生存为 94%，而无治疗组的 3年总体生存为 80%

（ P=0.03），肿瘤无进展比例在治疗组为 77%，而在无治疗组为 30%

（P<0.001）。故最近 Rajkumar研究者提出，对于此类患者应该直接诊断为 MM并按照MM开始治疗，而不是诊断为 SMM。这意味着在高危冒烟性骨髓瘤患者中再剥离出一部分进行治疗。会存在如何准确界定高危 SMM人群，会不会在MGUS

患者中造成过度治疗的问题，例如高免疫球蛋白以及高浆细胞代表疾病进展快，那么具有高危的肿瘤生物学特性是不是可以也应该早期治疗？如，患者出现一些高危细胞遗传学指证，17p-或是在 G显带中发现 13号染色体的缺失，这些患者是否应

该立即开始治疗，对于这一点尚未得到所有专家的认同，也无研究结果显示对这些患者早期干预后是否获益。目前仍建议在此类患者中密切随访，一旦出现器官功能的损害即开始治疗。本研究表明冒烟型多发性骨髓瘤血清游离轻链比值大于等于100是发展为有症状多发性骨髓瘤的高危因素。

杨天杰等对 60例临床确诊MM患者进行 IFE分析和 sFLC水平检测,并对结果进 行 分 析 。 结 果 血 清 IFE 分 析 结 果 :IgG 型 占 55.00%(33/60),IgA 型 占18.33%(11/60),IgM 型占 8.33%(5/60),单纯轻链型占 18.33%(11/60)。sFLC 检测结果:35例 κ 型 MM患者的 sFLC-κ水平和 25例 λ 型 MM患者的 sFLC-λ水平均高于正常参考范围(P<0.01);其 sFLC-κ/λ比值分别>1.65和<0.26。本研究结果与报道相一致。宋萍报道 37例初诊 LCMM患者(κ 型 17例,λ 型 20例),留取初诊及化疗后血清样

本,采用免疫比浊法测定 sFLC,同期测定患者 24 h尿轻链,分析 sFLC水平与 24 h尿轻链的相关性,及其与肾损害的关系。结果:全部患者初诊时 sFLC水平均明显升高,κ

型和 λ 型的中位值分别为 105.44 mg/L和 146.39mg/L。在诊断时和治疗后,血 sFLC

水平与尿轻链水平均无相关性。12例治疗后 24 h尿轻链水平正常患者中,8例血清游离轻链仍高于正常。应用 ROC分析评价初诊时轻链类型和水平与肾功能损害发生的关系结果显示,尿 λ轻链与肾功能损害发生相关,曲线下面积为 0.792(P=0.031)。结论:所有初诊 LCMM患者的 sFLC均明显增高。sFLC用于治疗反应的监测较尿轻链更加敏感。四、参考文献1.Mateos MV, Hernandez MT, Giraldo P, et al. Lenalidomide plus dexamethasone

for high-risk smoldering multiple myeloma[J]. N Engl J Med,2013, 369(5):438-447.

2. Rajkumar SV, Larson D, Kyle RA.Diagnosis of smoldering multiple myeloma[J].

N Engl J Med,2011, 365(5):474-475.

3. 杨天杰、龚斐然、周佳子等 。血清免疫固定电泳和游离轻链检测在多发性骨髓瘤的诊断价值。江苏医药，2016（3）

4. 宋萍、安志明、周小钢等。血清游离轻链的检测及其在轻链型多发性骨髓瘤中的临床意义。中国实验血液学杂志，2015（5）

类别：临床生化检验848017

The predictive value of the dynamic serum procalcitonin and C-reactive protein

levels on therapeutic effect and prognosis of pneumonia

OBJECTIVE: To assess the disease severity and prognosis by observing the kinetic

change of procalcitonin (PCT) and C-reactive protein (CRP) for pneumonia patients.

METHODS: The data were collected from January to December 2016 from The first

affiliated Hospital of Zhengzhou University. Demographic and clinical patient

information including age, length of hospital stay and number of comorbidities were

recorded. Blood samples were taken for CRP, PCT, white blood cell count (WBC).

Receiver Operating Characteristic (ROC) curve was used to verify each biomarker's

association with therapeutic effect and the prognosis of pneumonia. Multivariate Cox

regression analysis was used to identify which of these clinical parameters were

statistically associated with the pneumonia outcomes.

RESULTS: Three hundred and fifty patients were enrolled. ROC analysis showed serum

CRP5c setting at -4.524 had the highest efficiency in predicting the pneumonia outcomes.

CRP levels on day1, day3 were significant associated with pneumonia outcomes. PCT

levels on day5 (OR 1.134, 95%CI 1.056-1.219, p=0.001) was significantly associated

with pneumonia outcomes. Multivariate Cox regression showed there were week

correlation between CRP levels on day1 (OR 0.987, 95%CI 0.977-0.996, p=0.006), day3

(OR 1.008, 95%CI 1.002-1.014, p=0.013) and pneumonia outcomes. PCT levels on day5

(OR 1.134, 95%CI 1.056-1.219, p=0.001) was significantly associated with pneumonia

outcomes.

CONCLUSIONS: Serum initial CRP levels have moderate predictive value for

pneumonia prognosis. PCT levels on day5 during disease provided additional prognostic

information. The dynamic CRP and PCT levels may potentially be used in the future to

help monitor therapeutic effect and predict pneumonia outcome.

Keywords: dynamic serum PCT, CRP, predictive value, pneumonia outcome

1. Introduction

Diagnosis of pneumonia in critically ill patients is usually challenging. Signs and

symptoms with enormous heterogeneity, such as dyspnea, may be non-diagnostic or

atypical, chest X-ray results may be uncertain, also complications may be confounding

factors [1-3]. Thus, biomarkers of inflammation or infection, such as PCT and CRP, have

been proposed as a guide in the diagnostic process [4-6]. Elevated serum PCT and CRP

were associated with community-acquired pneumonia and ventilator-associated

pneumonia (VAP) [5, 7].

CRP is a well-established biomarker in many clinical settings, but has been traditionally

considered insufficient as a useful marker in the diagnosis of pneumonia. In fact, all

infections, stress reactions, autoimmunity and tumor disease can contribute to the

increase in serum CRP values [8].

PCT is a 116-amino acid long precursor of calcitonin, which is produced by the thyroid.

In sepsis, macrophages and the monocytic cells of the liver are involved in the synthesis

of PCT，which is elevated in sepsis [9, 10]. The degree of induction of PCT correlates

with the severity of systemic infection and the presence of organ dysfunction.

Several studies have reported controversial results in assessing the role of CRP and PCT

in the diagnosis of pneumonia in multiple elderly patients due to multiple confounding

factors [1, 11, 12]. The importance of serum CRP and PCT levels at diagnosis is well

established [5, 7, 13, 14]; however, its use remains unclear in the prediction of

pneumonia outcomes, and a dynamic approach of assessing biomarkers may provide

additional survival information. Markers of the inflammatory response and their kinetics

have been studied in the prediction of outcomes in sepsis [15] and VAP [16]. As reported

by Huang MY, et al, PCT clearance (PCTc) has been introduced in previous studies as a

tool for monitoring the changes of PCT levels during severe sepsis [17, 18]. Since PCTc

measures the relative changes in PCT to the baseline PCT, it is postulated to be a better

predictor of outcome.

Therefore, the hypothesis of this study is whether CRP and PCT levels and their

clearance could serve as prognostic biomarkers for critically ill pneumonia patients. The

aim of the present study was to evaluate the usefulness of CRP and PCT levels and their

clearance as biomarkers of prognosis for pneumonia in critically ill patients. We

undertook a study to assess the prognostic value of the kinetics of PCT, CRP and WBCs

in the outcome of pneumonia.

2. Materials and Methods

2.1. Study Design and Patient Population

This was a single-center, prospective observational study. Hospitalized Pneumonia

patients with a radiological confirmation were recruited. According to the WHO

indication, chest radiographs with alveolar or non-alveolar findings were initially

classified as of possible bacterial or of possible viral origin, respectively [19]. Then,

radiological findings were verified with results of the real-time PCR tests on blood

samples and nasopharyngeal swabs.

2.2. Measurement of Biomarkers

Followed our study design, WBC counts were measured as a part of routine tests. serum

CRP and PCT levels were measured on hospital days 1, 3, and 5 in patients. The blood

was drawn in vacuum tube filled with separation gel and centrifuged at 3500 rpm for 5

minutes, then biochemistry markers were analyzed within 30 minutes. Concentrations of

CRP were determined by an immunoturbidimetric assay. The diagnostic cut-off value of

CRP was set by manufacturer at 5 mg/L. PCT (ng/mL) levels were measured by electro

chemiluminescence immunoassay with a lower limit of detection of 0.02 ng/ml. PCTc on

day 3 (PCT3c-day 3) and that on day 5 (PCTc-day5) was calculated based on the

previously reported formula [18], (PCT day3/day5-PCT day1)/PCTday1×100%= PCTc

day3/day5 (%). CRP clearance (CRPc) were calculated referred to the PCTc formula.

PCTc on day3 and day5 were abbreviated as PCT3c and PCT5c, CRPc on day3 and day5

were abbreviated as CRP3c and CRP5c.

2.5 Statistical analysis and data management

Data were analyzed using SPSS v.17.0 software (SPSS Inc., Chicago, IL,

USA) for Windows. Pneumonia patients with different prognosis were compared using

one-way ANOVA test for continuous variables and chi-square (χ2) test for categorical

variables, adjusted for age and number of comorbidities. ROC curves were used to

evaluate the sensitivity and specificity of procalcitonin and CRP vs pneumonia prognosis.

Cut-off values were reported with the area under the curve (AUC) being given with its

95% confidence interval (CI). The cut-off value was calculated with the Youden index.

Multivariate cox regression models were then applied to identify parameters associated

with pneumonia prognosis. All p-values were two-tailed and were considered significant

for p < 0.05.

2.6 Outcomes

The primary endpoint was persistence of discomfort from infection at day 17. If the

discomfort disappeared completely, we defined the prognosis as Healing. If the

discomfort partly relieved, we defined the prognosis as improvement. The secondary

endpoint was the survival status of patients at day 28. Both endpoints were assessed by

seven medical students, blinded to the goal and design of the study, by conducting

standardized follow-up interviews by telephone at 28 days after baseline.

3. Results

3.1. Demographics and Clinical Presentations

Baseline characteristics of the patients included in the study are presented in Table 1.

This study included a total of 350 patients with a median age of 58.53 years (58.3%

males). Patients had a high burden of comorbidities including Congestive heart failure (n

=100), Poly trauma (n =77), Kidney diseases (n=47), cerebral hemorrhage and cerebral

infarction (n=39) and diabetes (n=30). Cough (n=268, 76.6%) and dyspnea (n =249,

71.1%) were the most frequent symptoms.

Table 1 Characteristics of the 350 patients of the study

Characters

Mean age ± SD (years) 58.53±19.13

Males (%) 204 (58.3)

Comorbidities

At least 1 comorbidity 274(78.29)

Diabetes Mellitus 30(8.57)

Liver disease 22(6.29)

Congestive heart failure 100(28.57)

cerebral hemorrhage and cerebral infarction 39(11.14)

History of Shock 17(4.86)

Chronic respiratory disease 22(6.29)

Poly trauma 77(22)

Kidney disease 47(13.43)

Neoplasia 26(7.43)

Signs and symptoms

Cough 268(76.6)

Chest pain 116(33.1)

Expectoration 168(48)

Dyspnea 249(71.1)

Chills 124(35.4)

Headaches 75(21.4)

Myalgia 79(22.6)

Crackles 114(32.6)

Fever 110(31.4)

Confusion 5(1.4)

Respiratory rate > 30/min 42(12)

Heart rate > 125/min 23(6.6)

3.2. ANOVA analysis for Clinical Factors and pneumonia

Table 2 Comparisons of biomarkers characteristics within different infection groups in pneumonia patients

Variable All subjects Bacterial Viral Fungal Undetermined

Males /Total (204 /350) (110/176) (44 /93) (20/32) (30/49)

Ages (years) 58.53±19.13 58.71±17.99 58.71±17.10 58.71±17.10 58.71±17.102

CRP1 (ng/L) 65.33±84.71 81.11±92.52*# 52.23±84.68* 30.43±30.57*# 53.55±74.03#

CRP3 (ng/L) 56.38±77.43 70.05±91.9*# 48.1±74.67* 37.16±32.74*# 41.55±47.47#

CRP3c 564.29±5085.35 156.31±904.83*# 575.76±3845.77#612.05±2298.79

*

1500.17±9885.52

*#

CRP5 (ng/L) 44.8±68.53 55.76±84.12*# 33.43±50.78# 38.14±43.05*# 34.28±44.72*

CRP5c 435.44±3509.55 141.25±910.69*454.46±3181.53

*290.29±948.01*

1174.87±6659.48

*

PCT1 (ng/mL) 1.82±7.08 2.25±5.83 0.89±2.95 0.9±2.08 2.06±11.74

PCT3 (ng/mL) 1.65±6.31 2.48±8.7 0.83±1.62 1.21±1.73 0.8±1.81

PCT3c 791.19±2653.76 759.01±2704.86 756.9±1959.071136.75±2638.8

6891.53±3192.05

PCT5 (ng/mL) 1.19±3.73 1.68±5.01* 0.57±0.94* 0.99±1.54 0.8±1.91

PCT5c 695.28±2463.03 635.57±2066.6 654.43±1871.631279.91±3286.5

2804.34±3455.5

WBC1 (109cells/μL) 10.31±8.08 11.54±10.23*§ 8.7±4.09* 9.71±5.68 8.9±3.7§

WBC3 (109cells/μL) 9.66±6.04 10.43±7.02 9.01±4.19 8±4.63 8.64±4.66

WBC5 (109cells/μL) 9.47±5.07 10.38±5.6* 7.47±3.61* 9.18±4.62 8.51±3.88

day (days) 17.70±6.80 17.71±5.60 16.29±5.56 20.32±15.30 17.69±6.75

*, §. The mean difference is significant at the 0.05 level.

WBCs, CRP and PCT levels on hospital days 1, 3, and 5 and their clearance were

compared in all groups. ANOVA analysis showed that CRP levels at

significant differences were found in terms of mean leukocyte counts for the

pneumonia patients infected with different pathogens. The average mean

value of these biomarkers comparison is reported in Table 2 and 3.

Table 3 Comparison of biomarkers characteristics within different prognosis

Variable All subjects improvement clinical cure death

n 350 48 264 38

CRP1 (ng/L) 65.33±84.71 104.51±106*§ 59.29±78.8* 57.06±81.29§

CRP3 (ng/L) 56.38±77.43 77.36±88.9§ 45±60.19*§ 109.13±128.45*

CRP3c 564.29±5085.35 778.37±4095.31*§ 547.78±5580.18§ 401.24±1242.37*

CRP5 (ng/L) 44.8±68.53 60.12±94.6* 34.02±52.22* 102.13±96.91*

CRP5c 435.44±3509.55 630.83±3262 401.61±3769.86 429.28±1207.59

PCT1 (ng/mL) 1.82±7.08 6.04±15.24*§ 1.04±4.1* 1.87±5.47§

PCT3 (ng/mL) 1.65±6.31 1.52±2.52 1.33±4.62* 4.07±14.49*

PCT3c 791.19±2653.76 93.12±479.15*§ 921.56±2882§ 774.07±2528*

PCT5 (ng/mL) 1.19±3.73 1.10±1.97* 0.76±2.04§ 4.26±9.26*§

PCT5c 695.28±2463.03 184.37±675.71* 664.19±2476.91* 1555±3454.35*

WBC1 (109cells/μL) 10.31±8.08 11.28±5.33 9.89±7.45 12.58±12.77

WBC3 (109cells/μL) 9.66±6.04 9.56±4.25 9.41±4.67 11.06±11.14

WBC5 (109cells/μL) 9.47±5.07 10.25±5.58 8.95±4.9 10.85±4.7

*, §. The mean difference is significant at the 0.05 level.

CRP levels at day1 and day3 were significantly different between the

different outcomes groups. Although the CRP levels at day1 and day3 were higher in the

improvement group than that in the other two groups, the CRPc at day3 were

significantly decreased compared to the other two groups.

Significant differences were found between different outcome groups with respect to

PCT levels on day1, day3 and day5. On day1, PCT levels in improvement group were

significantly higher than the other two groups. However, on day3 and day5 the

dramatically decrease in PCT levels often indicated a better prognosis including

improvement or healing, inversely the increase in PCT levels indicated the high risk of

death. PCT3c and PCT5c were significantly lower in the improvement group than the

other two groups, which showed PCT levels were dramatically reduced after treatment

despite the high initial PCT levels.

3.3 Correlation between PCT and CRP and their clearance

Assessment of correlation between biomarkers was performed by Spearman’s rank

correlation analysis. Table 4 showed correlations of CRP, PCT and their

clearance in the overall population. At baseline and day 3, we found no

significant correlations between PCT and CRP, however lower correlations were found at

day5 for PCT and CRP (R2=0.188, P<0.001).

Table 4 Correlation of biomarkers characteristics at different time

　 PCT1(ng/ml) PCT3(ng/ml) PCT5(ng/ml) PCT3c PCT5c

CRP1 (mg/L)R2=0.088P=0.103

CRP3 (mg/L)R2=0.066P=0.221

CRP3cR2=0.024P=0.66

CRP5(mg/L)R2=0.188P=0.000**

CRP5c

　 　

　 　 R2=0.043P=0.425

**. Pearson Correlation was used to test the correlation between biomarkers. Correlation is significant at the 0.01 level (2-tailed).

3.4 Prognostic performance of the studied biomarkers to predict pneumonia outcome

The ROC analysis showed that CRP levels on day3 (AUC 0.70, 95%CI 0.36-0.55) and

day5 (AUC 0.76, 95%CI 0.67-0.85), CRP3c (AUC 0.78, 95%CI 0.70-0.85) and CRP5c

(AUC 0.81, 95%CI 0.75-0.87) were significantly associated with the pneumonia

outcomes (Table 5). The cut-off value, with the best sensitivity and specificity

compromise, was set at -4.524 for CRP5c. On the other side, ROC analysis for

procalcitonin confirmed that this biomarker at baseline and day3 is not significantly

associated with pneumonia outcomes. However, significantly associated was found for

PCT levels on day5 (AUC 0.739, 95%CI 0.653-0.825), PCT3c (AUC 0.575, 95%CI

0.485-0.673) and PCT5c (AUC 0.657, 95%CI 0.567-0.746) with pneumonia outcomes

(Table 5 and Figure 1).

Table 5 Prognostic performance of Biomarkers in predicting pneumonia prognosis

Variable(s) AUC SE P 95%CIoptimal cut-

off value

CRP1 (mg/L) 0.455 0.05 0.373 0.358-0.552 　CRP3 (mg/L) 0.696 0.051 P<0.001 0.597-0.795 75.68

CRP3c 0.775 0.039 P<0.001 0.699-0.851 23.060

CRP5(mg/L) 0.764 0.046 P<0.001 0.674-0.853 39.76

CRP5c 0.81 0.029 P<0.001 0.753-0.867 -4.524

PCT1 (ng/mL) 0.579 0.048 0.112 0.485-0.673

PCT3 (ng/mL) 0.614 0.049 0.022 0.519-0.71 1.554

PCT3c 0.575 0.044 0.133 0.489-0.66

PCT5 (ng/mL) 0.739 0.044 P<0.001 0.653-0.825 1.025

PCT5c 0.657 0.046 0.002 0.567-0.746 51.802

a. Under the nonparametric assumption,

b. Null hypothesis: true area = 0.5

c ROC receiver operating characteristic, AUC area under the curve, CI confidence interval

Table 6 Multivariant Cox Regression of biomarkers characteristics with different prognosis

　 OR 95.0% CI Lower 95.0% CI Upper P

CRP1 0.987 0.977 0.996 0.006*

CRP3 1.008 1.002 1.014 0.013*

CRP3c 1 0.999 1.001 0.911

CRP5 1.007 0.998 1.015 0.113

CRP5c 1 0.999 1.001 0.798

PCT1 0.91 0.722 1.146 0.422

PCT3 0.986 0.898 1.082 0.767

PCT3c 1 1 1 0.305

PCT5 1.134 1.056 1.219 0.001*

PCT5c 1 1 1 0.968

Comorbidities 1.1 0.88 1.375 0.402

Age 0.989 0.971 1.007 0.213

3.5 Multivariate Cox regression exploring the association between biomarker levels and

pneumonia outcome

Table 6 summarizes the results of cox-regression analyses studying the association of

biomarker levels, biomarker’s clearance and pneumonia outcomes. Adjusting for the

number of comorbidities and age, analyses were performed according to day1, day3 and

day5 biomarker levels. For CRP, we found serum CRP levels on day1 (OR 0.987, 95%CI

0.977-0.996, p=0.006) and day3 (OR 1.008, 95%CI 1.002-1.014, p=0.013) were

significantly associated with pneumonia outcomes. For PCT, PCT levels on day5 (OR

1.134, 95%CI 1.056-1.219, p=0.001) was significantly associated with pneumonia

outcomes. In terms of CRPc and PCTc, no significant difference was found within the

different pneumonia outcome groups (P>0.05).

4 Discussion

In accordance with the current literature, the clinical characteristics of the patients

included in this study frequently had a comorbidity of respiratory disorders, diabetes

mellitus, congestive heart failure and cancer [20]. So far, most studies focused on the

diagnostic performance of serum biomarkers, especially CRP and PCT on the pneumonia

diagnosis [1, 5, 7, 11, 21]. Only very few research studied the predictive value of serum

biomarkers in the pneumonia outcomes [6, 14, 22, 23]. A dynamic approach to

biomarkers could capture the progression of disease and might be more effective in

evaluating pneumonia prognosis.

In this context, we observed serum CRP and PCT levels measured at different time points

after admission. The main findings of this study are threefold. First, circulating CRP and

PCT levels were significant different in the pneumonia patients infected with different

pathogens. Consistent with previous report[9, 10, 24], PCT is produced in large quantities

by many organs and cells in response to severe inflammation, especially bacterial

infection, while CRP served as a “inflammatory marker” increases independent of

infection type. CRP levels at day1 and day3 were significantly different between the

different outcome groups. The PCT levels on day1 and PCTc on day3 and day5 were

significant different between the three-outcome status.

Second, dynamic serum CRP levels, including CRP3, CRP5, CRP3c and CRP5c had

superior prognostic accuracy for the outcome prediction compared to serum PCT levels.

CRP5c at a cut-off value of -4.524 has the highest efficiency to predict the pneumonia

outcomes. While dynamic serum PCT levels had low or moderate prognosis value for the

pneumonia outcomes.

Third, consistent with the previous report[6], initial CRP and CRP3 levels were

independent prognostic predictors of clinical outcomes. PCT levels on day5 during

disease provided additional prognostic information. we found CRP levels on day1, day3

were significantly associated with pneumonia outcomes. PCT levels on day5 (OR 1.134,

95%CI 1.056-1.219, p=0.001) was significantly associated with pneumonia outcomes.

However, CRPc and PCTc were independent of pneumonia outcomes in cox regression

model. However, PCT has been used as a biomarker for initiating or terminating

antibiotic therapy in various clinical settings [25, 26]. The CRP level on day3 and PCT

level on day5 might reflect weather the antibiotic therapy was effective, and were

predictive for the pneumonia outcomes.

There were some limitations in our study. Firstly, the missing data for laboratory

biomarkers in some patients, potential classification bias in the etiologic diagnosis.

However, our evaluation has been done in a large study population even excluding

missing data. Secondly, no data were available about the number and the type of drugs

taken by each patient, particularly immunosuppressive drugs. Finally, the objects studied

usually combined with other diseases, which might affect the serum CRP and PCT levels.

But the complicated diseases were the true status for most elderly pneumonia patients.

Thus, further studies with a prospective design are needed to explore the influence of

each comorbidity on the biomarkers level and pneumonia prognosis.

5 Conclusions

This is a large and comprehensive study focused on the predictive value of serum

dynamic CRP, PCT levels and their clearance in pneumonia outcomes. The low

correlations between the two biomarkers and the only moderate prognostic accuracy calls

for a head-to-head trial comparing the ability of both markers to monitor the therapeutic

effect and to answer the question which marker is superior in the prognosis prediction.

Key messages

The dynamic serum CRP and PCT levels has moderate predictive value on therapeutic

effect and prognosis of pneumonia.

Declarations

Ethics, consent and permissions

The study protocol was approved by local ethics committee. All phases of the study were

carried out in conformity with the principles expressed in the Declaration of Helsinki.

Consent to publish

Written informed consent was obtained from the participant or guardian for children

according to Chinese law.

Availability of data and materials

All the preliminary data are available in supplement file 1.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

Shuren Guo did the data analysis and manuscription, Xiaohuan Mao and Xin Xie

contributed to the Data Curation, Liang Ming was responsible for the research activity

planning and execution. All authors read and approved the final version of the

manuscript.

Funding

This study was supported by the key research project of of Henan Education Science and

technology Foundation. (No.13A310622, NO.152102410067, NO162102310142 |

Acknowledgements

This research received no specific grant from any extra-institutional funding in the

public, commercial, or not for-profit sectors.

List of abbreviations

PCT: procalcitonin, CRP: C-reactive protein, WBC: white blood cell count, ROC:

Receiver Operating Characteristic curve, VAP: ventilator-associated pneumonia, PCTc:

PCT clearance, AUC: area under the curve, CRPc: CRP clearance.

Authors' information

1Department of Laboratory, The First Affiliated Hospital of Zhengzhou University,

Zhengzhou, Henan, People's Republic of China. Key Clinical Laboratory of Henan

province, Zhengzhou, Henan, People's Republic of China.

2 Department of Laboratory, Zhengzhou Yi He Hospital, Zhengzhou, Henan, People's

Republic of China. *corresponding author, mingliang3072@163.com

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类别：临床生化检验885213 基于纳米金原位合成生物传感器用于过氧化氢的检测

李身锋郑州大学第一附属医院检验科

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好，方法简单。1实验材料与仪器

μAUTOLAB-Ⅲ电化学工作站（荷兰 Ecochemie 公司），CHI660D电化学工作站（上海辰华仪器公司），B3200S-T 型超声清洗仪（美国 Branson 公司），pHS-

3C 型酸碱指示计（上海雷磁仪器公司），LIBROR AEL-200 电子天平（日本Shimadzu 公司），羧基化的多壁碳纳米管（MWCNT，直径 20 nm）购于成都有机化学研究所，氯金酸和硫堇购于 Sigma-Aldrich 公司，壳聚糖和辣根过氧化物酶购于上海生物工程有限公司，所用的氯化钠、醋酸钠、铁氰化钾、冰醋酸、无水乙醇均为分析纯级别试剂，实验用水为超纯水。2实验方法2.1 GNPs/MWCNTs–Chits 纳米复合物的制备

采用Wang Bo 等[19] 和Huang Haizhen等[20]利用壳聚糖（Chit）和氯金酸(HAuCl4)的简单绿色方法制备纳米金和MWCNTs的纳米复合物，具体方法如下：将 0.05 g的 Chit粉末分散在 10 ml的 1%(v/v)的醋酸缓冲液中，制备 0.5 wt%的壳聚糖溶液。取 1 ml制备好的 Chit溶液，然后加入 1 ml的MWCNTs，室温条件下超声分散，直到获得黑色均匀的混悬液。然后在制备的MWCNTs-Chit混悬液中加入 15

μL的 1 wt% HAuCl4溶液，混匀后在 80℃水浴中振荡孵育 1.5 h，然后取出，继续振荡至冷却到室温，然后转移到 4 ℃冰箱中保存。所制备的混合物通过扫描电子显微镜进行表征。2.2 过氧化氢传感器的制备过程将玻碳电极（直径为 3 mm）用 0.3 μm的Al2O3粉末抛光成镜面，然后依次在

超纯水、无水乙醇、超纯水中各超声洗涤 10 分钟，洗净晾干待用。然后在电极表面滴加 6 μL制备的GNPs-MWCNTs-Chit纳米复合材料，室温条件下晾干成膜。然后将GNPs-MWCNTs-Chits/GCE浸入含 3 mmol/L的硫堇醋酸缓冲液（HAc-

NaAc，0.1 mol/L，pH 6.0）里面，在 -0.5 V到+0.15 V (vs. Ag/AgCl)条件下以 50

mV/s 扫描 20圈，然后用超纯水，洗去非特异性吸附的硫堇溶液，室温条件下干燥。然后在电极表面滴加 3 μL的HRP和GNPs的混合液（v/v = 1:1），4 ℃干燥，即完成了HRP–GNPs/Thi/GNPs/MWCNTs–Chits/GCE的制备。该传感器性能的测定采用三电极系统，修饰过HRP的GCE电极为工作电极，铂丝电极为对电极，Ag/AgCl

电极为参比电极。3 结果和讨论3.1 不同组分在电极表面的电化学特性

图 3.1是GCE表面修饰不同成分在 0.1 mol/L的K3[Fe(CN)6]/K4[Fe(CN)6]（含0.1 mol/L KCl, pH 7.0）溶液中的循环伏安曲线。如图所示，和裸的玻碳电极相比，当电极表面仅修饰 GNPs-Chit（曲线 b）和MWCNTs-Chit（曲线 c）条件下，分别引起电流响应信号强度的降低和升高；而在电极表面修饰 GNPs/MWCNTs–Chits后（曲线 d），其产生的电流响应程度有了极大的提高，达到了最大值，这源于MWCNTs和 GNPs的协同作用，在 MWCNTs表面的 GNPs在一定程度上增加了MWCNTs的表面积，从而增强了MWCNTs对电流的放大作用。3.2 不同修饰电极的电化学特性

图 3.2为不同修饰电极在的 0.1 mol/L的 HAc-NaAc（pH 6.0）缓冲液中的循环伏安图像。如图所示，图 a是裸的玻碳电极，因GCE表面缺少电子介体，故其 CV

图像比较平滑；图 b为 GNPs/MWCNTs–Chit 修饰电极，图形较 a图曲线变宽，图像仍然平滑，这源于MWCNTs具有比较大的比表面积，能够放大电流信号，增加电子传递，但因缺乏电子介体，故没有出现氧化还原峰；而在电极聚合硫堇之后（图 c），可以看到电极表面出现一对氧化还原峰，电流响应程度较图 b有了极大的提高，这源于硫堇是一种电子介体，可以有效的存进电子传递，同时借助MWCNTs，使这种电子传递作用进一步增强；图 d为修饰了 HRP之后，因 HRP是蛋白质，可以阻碍电子的传递作用，从而导致电流较图 c出现下降，这也表明了HRP 已经成功的修饰到电极表面。3.3 传感器对过氧化氢的电化学响应

-0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2

-30

-25

-20

-15

-10

-5

0

5

10

15

E/V(vs.Ag/AgCl)

i / u

A

a

b

本研究所构建的传感器对过氧化氢响应的循环伏安图像如图 3.3所示，当传感器分别在空白的 HAc-NaAc溶液（曲线 a）和含有 2.0 mmol/L的过氧化氢溶液中（曲线 b）做循环伏安扫描时，氧化峰和还原峰都比较对称，并且曲线 b的氧化峰电流和曲线 a比起来基本没有变化，但是其还原峰电流较曲线 a明显增大，这说明在 HRP 催化过氧化氢的过程中，硫堇作为电子介体，促进电子的传递，出现了典型的HRP对过氧化氢的催化作用，表面修饰到电极表面的 HRP依然具备催化活性，制作的传感器对过氧化氢具有良好的信号响应能力。3.4 传感器的电化学响应特征、线性范围和检出限

在最佳的实验条件下，我们采用时间-电流曲线，通过在底物溶液中连续加入H2O2的方法对不同浓度 H2O2进行检测，并制作其电流和对应过氧化氢浓度的标准

曲线。如图 3.4所示，如图 1.7 (A)所示，随着过氧化氢的不断加入，该传感器对过氧化氢的响应明显，电流随着过氧化氢的加入，传感器的响应电流明显增大，且响应速度快。由图 1.7 (B)的标准曲线可知，图 B为在应用电位−0.23 V 条件下，测定的传感器的标准曲线，从曲线可知，过氧化氢浓度在 5×10−7 mol/L ~ 1.5×10−3 mol/L

范围内和响应电流呈线性关系，相关系数 r 为 0.9985，检测下限为 3.75×10−8

mol/L。表明该传感器具有较好的检测灵敏度。结论本研究中我们利用壳聚糖对氯金酸的还原作用，实现纳米金在MWCNTs表面

的原位合成，并对其进行表征，然后利用所制备的 GNPs-MWCNTs纳米复合物构建H2O2生物传感器，结果显示，该传感器综合利用了MWCNTs和GNPs的优点，对H2O2电化学响应良好。

类别：临床生化检验905571血清 MMP-8、TIMP-1和MMP-8/TIMP-1水平在急性冠脉综合征病情和预后评估

的作用分析苏佳

河南省中医院（河南中医学院第二附属医院）检验科 450003

【摘要】目的 探讨血清基质金属蛋白酶-8(MMP-8)、基质金属蛋白酶抑制剂-

1(TIMP-1)和MMP-8/TIMP-1水平在急性冠脉综合征（ACS）病情和预后评估的作用。 方法 选取 2014年 1月至 2016年 8月于我院行冠脉造影检查者 160例，其中105例确诊 ACS患者为 ACS组，其余 55例健康者为对照组。检测比较两组血清MMP-8和 TIMP-1水平、MMP-8/TIMP-1和 LVEF。ACS组均常规进行治疗，并在PCI 术后 7d再次检测血清MMP-8、TIMP-1和MMP-8/TIMP-1水平及 LVEF，出院后随访至少 1年，统计 ACS组生存率。分析 ACS患者血清MMP-8、TIMP-1和MMP-8/TIMP-1水平与 LVEF和 1年生存率的关系，及其预测 1年生存预后的价值。结果 与对照组比较，ACS组血清MMP-8、TIMP-1和MMP-8/TIMP-1水平均升高，LVEF 则降低（P＜0.05）。与术前比较，ACS组术后 7d血清MMP-8、TIMP-1和MMP-8/TIMP-1水平均降低，LVEF 则升高（P＜0.05）。ACS组随访 1年生存率为85.71%（ 90/105）。与存活患者比较，ACS 组死亡患者围术期血清 MMP-

8、TIMP-1和MMP-8/TIMP-1水平均升高，LVEF 则降低（P＜0.05）。Spearman

相关分析结果显示， ACS患者血清 MMP-8、TIMP-1和 MMP-8/TIMP-1水平与LVEF和 1年生存率均呈负相关（P＜0.05）。ROC曲线分析结果显示，ACS患者血清MMP-8、TIMP-1和MMP-8/TIMP-1水平预测其 1年生存预后的准确性均较高，其中以术后 7d血清 MMP-8/TIMP-1水平预测其 1年生存预后的准确性最高。结论 血清MMP-8、TIMP-1和MMP-8/TIMP-1水平与 ACS患者 LVEF和 1年生存率均密切相关，可能作为其病情和预后评估的参考指标。

【关键词】基质金属蛋白酶-8；基质金属蛋白酶抑制剂-1；MMP-8/TIMP-1；

急性冠脉综合征；病情；预后Value of serum MMP-8, TIMP-1 and MMP-8 / TIMP-1 levels evaluating disease severity

and prognosis of acute coronary syndrome

[Abstract] Objective: To investigate the value of serum metalloproteinase-8 (MMP-8), matrix metalloproteinase-1 (TIMP-1) and MMP-8 / TIMP-1 levels evaluating disease severity and prognosis of acute coronary syndrome (ACS). Methods: 160 cases of persons had coronary angiography in our hospital from January 2014 to August 2016 were selected. Among them, 105 patients diagnosed as ACS were enrolled in ACS group, and the other 55 healthy subjects were enrolled in control group. Serum MMP-8 and TIMP-1 levels, MMP-8 / TIMP-1 and LVEF of the 2 groups were detected. ACS group had PCI treatment and 7 days after PCI serum MMP-8 and TIMP-1 levels, MMP-8 / TIMP-1 and LVEF of ACS group were detected. The 1 year survival rate of ACS was counted. The relationship between serum MMP-8, TIMP-1 and MMP-8 / TIMP-1 levels with LVEF and 1-year survival in ACS patients were analyzed, and value of serum MMP-8, TIMP-1 and MMP-8 / TIMP-1 levels predicting the1 year survival prognosis were analyzed. Results: Compared with the control group, serum MMP-8, TIMP-1 and MMP-8 / TIMP-1 levels in ACS group were increased, while LVEF in ACS group was decreased (P＜0.05). Compared with preoperative, 7 days after PCI serum MMP-8, TIMP-1 and MMP-8 / TIMP-1 levels in ACS group were decreased, while in the same time LVEF in ACS group was increased (P＜0.05). The 1-year follow-up rate in ACS group was 85.71% (90/105). Compared with survivors, serum MMP-8, TIMP-1 and MMP-8 / TIMP-1 level of the dead patients in ACS group were increasedm, while LVEF of the dead patients in ACS group was decreased (P＜0.05). Spearman correlation analysis showed that serum MMP-8, TIMP-1 and MMP-8 / TIMP-1 levels were negatively correlated with LVEF and 1-year survival rates in ACS patients (P <0.05). ROC curve analysis showed that accuracy of serum MMP-8,TIMP- 1 and MMP-8 / TIMP- 1 level predicting the 1-year survival prognosis were high, in which the accuracy of 7 days after PCI serum MMP-8 / TIMP- 1 level predicting the 1-year survival prognosis was the highest. Conclusion: Serum MMP-8, TIMP-1 and MMP-8 / TIMP-1 levels in ACS patients are closely related to LVEF and 1-year survival rates, which may be used as reference indexes for disease severity and prognosis evaluation.[Key words] matrix metalloproteinase-8; matrix metalloproteinase inhibitor-1; MMP-8 / TIMP-1; acute coronary syndrome; disease severity; prognosis冠心病为临床常见心脏疾病，而急性冠脉综合征（acute coronary

syndrome，ACS）为冠心病严重类型，其起病急，病情发展快，死亡率高，预后情况差[1,2]。改善 ACS疗效和预后是目前急需解决的医疗问题。临床已有多个研究证实基质金属蛋白酶-8(MMP-8)和基质金属蛋白酶抑制剂-1(TIMP-1)与冠心病和ACS密切相关[3-5]。因此，本研究假设联合检测分析MMP-8和 TIMP-1可能更有利于对ACS病情和预后评估。因此，本研究检测了ACS患者的MMP-8、TIMP-1

和MMP-8/TIMP-1，分析了其与患者心功能及 1年生存预后的关系及其对患者预后的早期预测价值，旨在为ACS疗效和预后的改善提供依据，研究结果如下。1资料与方法1.1一般资料 选取 2014年 1月至 2016年 8月于我院行冠脉造影检查者 160例，其中 105例临床影像学、实验室指标和冠脉造影检查确诊的ACS患者为ACS组，其余 55例检查结果正常的健康者为对照组，两组均无心脏疾病史、无其他严重内外科疾病，精神正常，非妊娠期或哺乳期。两组入组前均知情同意并签署知情同意书，研究符合伦理学标准。ACS组和对照组性别比、年龄、身体质量指数等基线资料比较差异均无统计学意义（P＞0.05），具有可比性，见表 1。

表 1两组基线资料比较组别 例数 性别比（男/

女）年龄(岁) 身体质量指数

（kg/m2）ACS组 105 65/40 63.56±8.78 21.19±3.56

对照组 55 39/16 63.41±9.22 21.42±3.75

统计量 ⅹ2=1.385 t=0.101 t=0.381P ＞0.05 ＞0.05 ＞0.05

1.2治疗方法 ACS组均常规进行经皮冠状动脉介入（PCI），具体操作同参考文献[6]。围术期采用阿司匹林、氯吡格雷、低分子肝素、阿托伐他汀、血管紧张素转换酶抑制剂、硝酸酯等药物治疗，具体用药方法、计量和疗程均严格按照相关说明书进行，患者均严格按照医嘱和护士要求配合完成相关治疗。出院后患者均门诊或电话随访至少 1年，每 3个月随访 1次，随访截止日期为 2017年 8月 31日，期间无患者失访，随访率 100.00%（105/105）。

1.3 观察指标和检测方法 （1）分别两组入组次日均取空腹静脉血 5ml进行血清基质金属蛋白酶-8(MMP-8)和基质金属蛋白酶抑制剂-1(TIMP-1)水平的检测，将获得的血液标本置于 EDTA抗凝管中，上离心机，以 3500r/min转速、3cm离心半径离心 10min，待分层后取上层血清冷藏于-20℃低温冰箱待测，检测均采用日立7010 型全自动生化分析仪，相关试剂盒均由上海沪震实业有限公司提供，操作严

格按照仪器和试剂盒说明书要求进行，并计算MMP-8/TIMP-1比值。（2）并在两组入组后进行左室射血分数（LVEF）的检测，检测仪器为HDI 3000彩色超声诊断仪进，探头频率 2.5MHz，取平卧位，保持呼吸平静，常规获取标准各个心脏切面图像，计算 LVEF（LVEF=每搏量/心室收缩末期容积）。（3）ACS组 PCI术后 7d

再次取空腹静脉血 5ml检测血清MMP-8、TIMP-1和MMP-8/TIMP-1水平及LVEF，具体检测操作同术前。（4）统计患者随访至少 1年的生存预后情况，并统计患者 1年生存率。

1.4统计学方法 采用 SPSS22.0软件，计数资料比较采用卡方检验，计量资料均符合正态分布并采用两独立样本均数 t检验进行组间比较，采用Kaplan Meier生存曲线分析ACS组随访 1年的生存预后情况，Spearman相关分析法分析ACS患者血清MMP-8、TIMP-1和MMP-8/TIMP-1水平与 LVEF和 1年生存率的关系，采用受试者操作特性曲线（ROC）分析ACS患者血清MMP-8、TIMP-1和MMP-8/

TIMP-1水平预测其 1年生存预后的价值，P＜0.05为差异有统计学意义。2结果2.1两组血清MMP-8、TIMP-1和MMP-8/TIMP-1水平以及 LVEF比较与对照组比较，ACS组血清MMP-8、TIMP-1和MMP-8/TIMP-1水平均升高，

LVEF 则降低，差异有统计学意义（P＜0.05）。与术前比较，ACS组术后 7d血清MMP-8、TIMP-1和MMP-8/TIMP-1水平均降低，LVEF 则升高，差异有统计学意义（P＜0.05），见表 2。

表 2两组血清MMP-8、TIMP-1和MMP-8/TIMP-1水平以及 LVEF比较组别 时间 例数 MMP-

8（ng/mL） TIMP-1（ng/mL）

MMP-8/TIMP-1

LVEF（%）ACS组 术前 105 61.53±8.66* 136.25±15.78* 0.45±0.09* 42.56±6.55*

术后 7d 105 42.88±6.85*# 115.09±11.72*# 0.37±0.07*# 58.22±8.72*#

对照组 入组次日

55 26.58±5.78 80.26±9.25 0.32±0.05 65.78±5.65

注：与对照组比较，*P＜0.05；与同组术前比较，#P＜0.05。

2.2 ACS组随访 1年生存预后分析ACS组随访 1年 15例患者死亡，存活患者 90例，随访 1年生存率为

85.71%（90/105），见图 1。

图 1ACS组随访 1年生存预后的Kaplan Meier生存曲线分析2.3 ACS组不同生存预后患者的血清MMP-8、TIMP-1和MMP-8/TIMP-1水平

及 LVEF比较与存活患者比较，ACS组死亡患者围术期血清MMP-8、TIMP-1和MMP-8/

TIMP-1水平均升高，LVEF 则降低，差异有统计学意义（P＜0.05），见表 3。表 3 ACS组不同生存预后患者的血清MMP-8、TIMP-1和MMP-8/TIMP-1水平及

LVEF比较时间 生存预

后例数 MMP-

8（ng/mL） TIMP-1（ng/mL） MMP-8/

TIMP-1LVEF（%）

术前 死亡 15 72.51±8.95 155.26±16.53 0.47±0.06 38.25±6.16

存活 90 59.70±7.52 133.08±13.86 0.45±0.03 43.28±6.58t 5.942 5.586 2.015 2.764P ＜0.05 ＜0.05 ＜0.05 ＜0.05

术后7d

死亡 15 56.25±7.22 130.58±12.85 0.43±0.05 49.25±8.22

存活 90 40.65±5.76 112.51±10.28 0.36±0.04 59.72±6.52t 9.355 6.075 6.048 5.540P ＜0.05 ＜0.05 ＜0.05 ＜0.05

2.4 ACS患者血清MMP-8、TIMP-1和MMP-8/TIMP-1水平与 LVEF和 1年生存率的关系分析

Spearman相关分析结果显示， ACS患者血清MMP-8、TIMP-1

和MMP-8/TIMP-1水平与 LVEF和 1年生存率均呈负相关（MMP-8：r=-0.878，-

0.852，；TIMP-1：r=-0.863，-0.859，；MMP-8/TIMP-1：r=-0.882，-0.845，P＜0.05）。

2.5 ACS患者血清MMP-8、TIMP-1和MMP-8/TIMP-1水平预测其 1年生存预后的价值分析

ROC曲线分析结果显示，ACS患者血清MMP-8、TIMP-1和MMP-8/TIMP-1

水平预测其 1年生存预后的准确性均较高，其中以术后 7d血清 MMP-8/TIMP-1水平预测其 1年生存预后的准确性最高，见表 4、图 2和图 3。表 4 ACS患者血清MMP-8、TIMP-1和MMP-8/TIMP-1水平预测其 1年生存预后的

价值时间 因子 临界值 敏感度 特异度 准确性术前 MMP-8 72.06ng/mL 66.67% 90.00% 86.67%

TIMP-1 153.89ng/mL 60.00% 88.89% 84.76%MMP-8/TIMP-1

0.4780.00% 94.44% 92.38%术后

7dMMP-8 55.78ng/mL 86.67% 94.44% 93.33%TIMP-1 129.65ng/mL 80.00% 91.11% 89.52%MMP-8/TIMP-1

0.4293.33% 97.78% 97.14%

图 2 PCI术前ACS患者血清MMP-8、TIMP-1和MMP-8/TIMP-1水平预测其 1年生存预后的 ROC曲线

图 3 PCI术后 7dACS患者血清MMP-8、TIMP-1和MMP-8/TIMP-1水平预测其 1年生存预后的 ROC曲线

3讨论冠心病是目前威胁人类健康和生命安全的重要疾病，且近年来随着生活方式、

饮食结构的改变以及人口老龄化的不断发展，冠心病的发病率不断增加，其治疗干预为目前临床医师关注的重点和难点。急性冠脉综合征为冠心病严重类型，患者病情严重，预后情况差[7,8]。本研究亦关注了急性冠脉综合征患者的心功能和预后情况，本研究结果显示，急性冠脉综合征患者的 LVEF多在 45%以下，心功能低下，且其治疗后 1年的死亡率高达 10%以上，患者预后情况差，急需改善。急性冠脉综合征患者病情发生发展常伴随着多个生物标志物的异常[9,10]。其

中基质金属蛋白酶是已被证实与冠心病密切相关因子，其中的基质金属蛋白酶 8属间质胶原酶，与感染具有一定的相关性，平时储存在中性粒细胞中，而当体内趋化因子刺激和炎症反应情况下可释放，造成其血液浓度的明显升高[11-13]。因此，基

质金属蛋白酶 8可能通过参与炎症反应而影响机型冠脉综合征的发生发展，可能用于其病情和预后评估。基质金属蛋白酶抑制剂可调节基质金属蛋白酶活性，而基质金属蛋白酶抑制剂与基质金属蛋白酶的失衡与炎症反应等病理过程密切相关[14-

16]。因此，联合评估基质金属蛋白酶抑制剂与基质金属蛋白酶平衡状况可能更好地评估急性冠脉综合征等炎症相关疾病状况。然而目前关于此方面研究较少，需进一步研究证实。因此，本研究分析了急性冠脉综合征患者基质金属蛋白酶抑制剂 1和基质金属

蛋白酶 8以及其比值与其心功能和预后的关系。本研究结果显示，急性冠脉综合征患者基质金属蛋白酶抑制剂 1和基质金属蛋白酶 8水平以及其比值均明显高于健康人群，进一步证实了基质金属蛋白酶抑制剂、基质金属蛋白酶以及两者的平衡状况可能与急性冠脉综合征相关。而本研究中随访 1年死亡患者围术期血清MMP-

8、TIMP-1和MMP-8/TIMP-1水平均升高，LVEF 则降低，提示其围术期血清MMP-8、TIMP-1和MMP-8/TIMP-1水平与心功能和 1年生存预后可能相关。进一步的 Spearman相关分析结果显示，ACS患者血清MMP-8、TIMP-1和MMP-8/

TIMP-1水平与 LVEF和 1年生存率均呈负相关，可能用于其病情评估和预后早期预测。更进一步的 ROC曲线分析结果显示，ACS患者血清MMP-8、TIMP-1和MMP-8/TIMP-1水平预测其 1年生存预后的准确性均较高，其中以术后 7d血清

MMP-8/TIMP-1水平预测其 1年生存预后的准确性最高。对ACS患者基质金属蛋白酶抑制剂与基质金属蛋白酶平衡状况进行评估可能有利于指导临床早期进行干预，以改善病情和预后，而通过开放维持基质金属蛋白酶抑制剂与基质金属蛋白酶平衡状况的药物应用于临床治疗可能为改善急性冠脉综合征病情和预后的有效途径之一，此方向仍需进一步研究证实。综上所述， 血清MMP-8、TIMP-1和MMP-8/TIMP-1水平与ACS患者 LVEF

和 1年生存率均密切相关，可用于其病情和预后评估，指导临床干预以期改善病情和预后。

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[4]江琼,庄和,郑行春,等.原发性高血压不同危险分层患者 RDW-CV、MMP-

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类别：临床生化检验899613 同型半胱氨酸与肝炎严重程度的相关性研究

王瑞锋平煤神马医疗集团总医院检验科

【摘要】目的 探讨同型半胱氨酸与肝炎严重程度间的相关性。方法 以 2013年 1

月至 2016年 10月我院收治的 112例慢性乙型肝炎患者为研究对象，其中轻度肝炎 21例，中度肝炎 72例，重度肝炎 19例，取同期来本院做健康体检的人员 30

名作为正常组。检测两组血清生物化学指标丙氨酸转氨酶（ALT）、门冬氨酸转氨酶（AST）、总胆红素（TBIL）、胆碱酯酶（CHE）及同型半胱氨酸（Hcy）浓度。比较两组间及患者组不同病情程度组间上述各指标的血清水平差异，并对患者组内生化指标与同型半胱氨酸的相关性进行分析。结果 患者组的平均血清同型半胱氨酸水平为（24.72±4.34）μmol/L，正常组为（8.34±2.35）μmol/L，组间比较患者组高于正常组，差异具有统计学意义(P<0.05)。轻度、中度、重度患者组的平均血清同型半胱氨酸水平分别为（9.54±2.63）、（23.41±4.62）与（35.25±63.41）μmol/L，三组间存在显著性差异，具有统计学意义(P<0.05)，轻度组<中度组<重度组(P<0.05)。两组间各生化指标比较差异有统计学意义(P<0.05)，患者组血清ALT、AST、TBIL水平高于正常组，CHE水平则低于正常组。其中ALT、AST、TBIL与Hcy呈正相关，CHE与Hcy呈负相关(P<0.05)。结论 乙型肝炎患者机体Hcy水平变化反映了肝脏的受损情况，可将Hcy 持续升高视为肝炎进展的危险因素，为临床诊断、治疗和预后提供帮助。【关键词】同型半胱氨酸；Hcy；肝炎；相关性 

同型半胱氨酸(homocysteine，Hcy) 是一种对机体有害的含硫氨基酸，为蛋氨酸代谢过程中重要中间产物[1]。已有完整资料证实高同型半胱氨酸会大幅增加冠心病、外周血管疾病及脑血管疾病的发病风险，因此被做为心脑及外周血管疾病

的重要危险因素及预测指标[2]。肝脏是氨基酸代谢的重要器官，当乙型肝炎病毒侵犯机体造成肝脏损伤时，会导致氨基酸代谢紊乱，发生血清氨基酸谱的改变。本研究对部分慢性乙型肝炎患者的同型半胱氨酸水平进行检测分析，以探讨肝炎患者同型半胱氨酸浓度的变化在肝脏病理变化的作用，为此类患者的临床治疗、预后及监测提供参考。1资料与方法1.1 一般资料 以 2013年 1月至 2016年 10月我院收治的 112例慢性乙型肝炎患者为研究对象。诊断与分组参照 2000年 9月中华医学会传染病与寄生虫病学分会、肝病学分会在西安联合修订的《病毒性肝炎防治方案》以及 2010年中华医学会肝病学分会、中华医学会感染病学分会联合修订的《慢性乙型肝炎防治指南》。男 79例，女 33例；年龄 21～54岁，平均(41.78±3.41)岁；病程 1.5~13年，平均(7.15±2.48)年；轻度肝炎 21例，中度肝炎 72例，重度肝炎 19例。取同期来本院做健康体检者 30名作为正常组，其中男 21名，女 9名；年龄 24～52

岁，平均(42.32±3.35)岁。两组间性别、年龄差异无统计学意义（P>0.05），具可比性。1.2方法 清晨空腹用真空采血管取静脉血两管，每管 5mL，3000r/min离心15min，取上清，放置于 5 ℃冰箱里备用。血清生物化学指标丙氨酸转氨酶（ALT）、门冬氨酸转氨酶（AST）、总胆红素（TBIL）、胆碱酯酶（CHE）采用 SIEMENS ADVIA全自动生化分析仪进行检测,试剂由利德曼生物有限公司提供，质控品为专用。1.3统计学方法 应用 SPSS20.0统计软件进行数据处理，计量资料以 x±s表示，两组间比较采用 t检验，多组间比较采用 F检验，组间两两比较采用 LSD-t检验，两连续变量关系采用直线相关分析法，P<0.05为差异有统计学意义。2结果

2.1 乙型肝炎患者组与正常组 Hcy水平比较 患者组的平均血清Hcy水平高于正常组，差异具有统计学意义(P<0.01)。见表 1.

表 1 乙型肝炎患者组与正常组Hcy水平比较(ⅹ±s)

组别 例数 Hcy(μmol/L)

患者组 112 24.72±4.34正常组 30 8.34±2.35

注:t=19.871,P=0.000

2.2 乙型肝炎患者Hcy水平与病情程度关系 轻度组、中度组、重度组的平均血清Hcy水平差异有统计学意义(P<0.05)；两两比较结果显示，三组间血清Hcy水平高低顺序为轻度组<中度组<重度组(P<0.05)。见表 2.

表 2 乙型肝炎患者Hcy水平与病情程度关系(ⅹ±s)

组别 例数 Hcy(μmol/L)

轻度组 21 9.54±2.63

中度组 72 23.41±4.62

重度组 19 35.25±63.41

注：F=19.746,P=0.000

2.3 患者组和正常组各生化指标检测结果比较　两组间各生化指标比较差异有统计学意义(P<0.05)，患者组血清ALT、AST、TBIL水平高于正常组，CHE水平则低于正常组(P<0.01)。见表 3.

表 3 患者组合和正常组各生化指标检测结果比较(ⅹ±s)

组别 例数 ALT（U/L） AST（U/L） TBIL(μmol/L)CHE（KU/

L）患者组 112

299.41±26.52

248±24.10

257±39.132.74±0.6

3

正常组 30 21.71±3.4623.41±3.5

98.10±2.05

6.74±1.84

t - 21.041 24.320 28.431 4.535P - 0.000 0.000 0.000 0.000

2.4患者组生化指标与Hcy相关性分析 ALT、AST、TBIL及CHE均与Hcy显著相关，其中ALT、AST、TBIL与Hcy呈正相关，CHE与Hcy呈负相关(P<0.05)。见表 4.

表 4 患者组生化指标与Hcy相关性分析结果ALT AST TBIL CHE

r 0.285 0.412 0.452 -0.386P 0.012 0.008 0.010 0.014

3 讨论3.1 Hcy学名为 2-氨基 4-巯基丁酸，是蛋氨酸代谢产生的一种含硫氨基酸，80%在血中通过二硫键与蛋白质结合，只有少部分同型半胱氨酸以游离状态存在于血液循环，为反应性血管损伤氨基酸[3]。目前临床已将Hcy 公认为是动脉粥样硬化的主要危险因子，将其用于心脑血管疾病的诊断[4]。研究发现，转甲基反应和Hcy 代谢的重要场所，Hcy 主要在肝脏内合成和分解，当肝脏功能受损时，势必会影响到甲硫氨酸循环和Hcy的代谢[5]。本研究结果显示，患者组的平均血清Hcy水平较正常组显著增高，且随着疾病严重程度的增加，Hcy水平亦稳步提升。Hcy 主要来源于饮食摄取的蛋氨酸，是蛋氨酸脱甲基后的产物。当肝功能受损时，氨基酸代谢障碍，Hcy在细胞内蓄积，导致血清水平升高，加重肝细胞负担，进一步加重代谢负担，进而加速肝脏疾病恶化的进程。有文献报道，肝硬化等慢性肝病患者可出现高Hcy血症，且Hcy水平与肝损害程度成正比[6-7]。

3.2 当细胞严重受损时，线粒体遭到破坏，存在于其中的酶释放出来，而大部分肝功能酶学指标都存在于细胞线粒体内，因此酶学指标可以反映肝炎病情[8]。ALT、AST、TBIL、CHE均为肝功能酶学重要指标。TBIL是反映肝脏代谢功能的重要指标[9-10]。本组研究结果显示，两组间各生化指标比较差异有统计学意义(P<0.05)，患者组血清ALT、AST、TBIL水平高于正常组，CHE水平则低于正常组。TBIL是反映肝脏代谢功能；CHE 主要由肝细胞合成，其水平高低与肝细胞受损程度相关，当肝细胞变性坏死时，会导致CHE合成减少，病情越严重，CHE

下降幅度越大[11-12]。与本研究中结果相一致。此外，ALT、AST、TBIL及CHE均与Hcy显著相关，其中ALT、AST、TBIL与Hcy呈正相关，CHE与Hcy呈负相关(P<0.05)。提示在肝炎患者中，随着ALT、AST和 TBIL的升高，Hcy水平亦随之上升，而随着CHE水平的下降，Hcy处于升高的状态。Hcy在肝脏代谢中起着非常重要的作用，其水平的变化像肝功能酶学指标一样直接反映了肝脏的储备功能、蛋白质代谢功能和肝细胞的损伤情况，因此可将其视为检测乙型肝炎患者肝功能的良好指标。综上所述，乙型肝炎患者机体Hcy水平变化与病情具有相关性，反映了肝脏

的受损情况，可将Hcy 持续升高视为肝炎进展的危险因素，为临床诊断、治疗和预后提供帮助。 参考文献[1]张琳,吴秀艳,薛晓琳,等. 基于现代文献的肝硬化中医证候及证候要素分布特点的研究[J]. 北京中医药大学学报,2012,35(11):747-751.

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类别：临床实验室生物安全管理及分子诊断技术与临床应用879770MicroRNA-10a targets CHL1 and promotes cell growth, migration and invasion

in human cervical cancer cells

Mei-Jing Long a,1 , Fu-Xia Wu b,1 , Pu Li c,1 , Min Liu b , Xin Li b , Liang Ming a,*a Department of Clinical Laboratory, the First Affiliated Hospital of Zhengzhou

University, Chinab Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical

University, Tianjin, Chinac Tianjin Central Hospital of Gynecology Obstetrics, Tianjin, China

Correspondence to: Dr. Liang Ming, Department of Clinical Laboratory, the First

Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, P.R. China

E-mail: mingliang@zzu.edu.cn1 These authors contributed equally to this work.

Abbreviations: ASO, antisense oligonucleotides; cDNA, complement DNA; CHL1, cell

adhesion molecule with homology to L1CAM (close homolog of L1); ECM, extracellular

matrix; EGFP, enhanced green fluorescence protein; GAPDH, glyceraldehyde-3

phosphate dehydrogenase; L1CAM, L1 family of cell adhesion molecule; miR-10a,

microRNA-10a; miRNA, microRNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide; NC, negative control; ORF, open reading frame; RFP, red

fluorescence protein; RT-PCR, reverse transcription polymerase chain reaction; siRNA,

small interfering RNA; UTR, untranslated region

ABSTRACT

MicroRNAs (miRNAs) play an important role in cancer initiation, progression and

metastasis by regulating their target genes. Here, we found microRNA-10a (miR-10a) is

upregulated in human cervical cancer and promotes the colony formation activity,

migration and invasion of HeLa and C33A cells. Subsequently, CHL1 is confirmed as a

target of miR-10a and is negatively regulated by miR-10a at mRNA and protein levels.

Futhermore, knockdown of CHL1 expression results in increased colony formation

activity, migration and invasion. Finally, overexpression of CHL1 lacked the 3’UTR

abolished the effects of miR-10a. Our results may provide a strategy for blocking tumor

metastasis.

Keywords: microRNA-10a; CHL1; cervical cancer; migration; invasion.

1. Introduction

MicroRNAs (miRNAs) are evolutionarily conserved, small noncoding endogenous

RNA molecules that function as posttranscriptional gene regulators by pairing with the 3’

untranslated regions (3’UTR) of specific target messenger RNAs (mRNAs) to suppress

translation or induce mRNA degradation [1,2]. Emerging evidence has revealed that

miRNAs are involved in various physiological and pathological processes, such as

inflammation, cell cycle regulation, differentiation, tumorigenesis, apoptosis and invasion

[3-6]. In addition, several studies have demonstrated that many miRNAs function as

oncogenes or antioncogenes and play key roles in cancer initiation and progression by

regulating their target genes [7-15], which are involved in essential steps during cancer

progression, including cell invasion, cell migration, and metastasis [16,17]. However, the

role of miRNAs in mediating tumor metastasis has only been recently addressed and still

remains largely unexplored. It is believed that knowledge about the relationship between

target genes and miRNAs is important for understanding the regulatory mechanism of

miRNAs in animal development as well as in tumor invasion and metastasis.

Neural cell adhesion molecules of the immunoglobulin (Ig) superfamily play important

roles in the development and regeneration of the nervous system [18]. The L1 family of

cell adhesion molecules (L1-CAMs) is comprised of L1, Close Homolog of L1 (CHL1,

L1CAM2), NrCAM, and Neurofascin, which are structurally related transmembrane

proteins in vertebrates. Recently, various studies have demonstrated that L1CAM is

overexpressed in human carcinomas, including breast cancer [19], colon cancer [20,21],

ovarian and endometrial tumors [22], gallbladder carcinoma [23], pancreatic ductal

adenocarcinoma [24], and non-small cell lung cancer [25], where it participates in

metastasis and progression and is associated with poor prognosis. The function of the

soluble form of L1CAM in promoting breast cancer cell adhesion to the extracellular

matrix (ECM) and cell migration has been shown [19]. However, very few studies that

focus on the expression and function of CHL1 in human cervical carcinomas have been

reported. Senchenko et al. reported that CHL1 was silenced to facilitate in situ tumor

growth as a putative tumor suppressor [26]. In our research, we have taken great interest

in the expression and function of CHL1 in human cervical carcinomas. Cervical cancer is

the third most common cancer in the female population. The disease and its financial

impact are significant. Recently, we have revealed several miRNAs that may act as

oncogenes or tumor suppressors in human cervical cancer, such as oncogenic miR-20a

[27] and miR-19a/b [28], and tumor suppressors miR-214 [29-31] and miR-372 [32]. In

characterizing the expression and function of CHL1 in human cervical carcinomas,

miRNAs may serve as one of the potential regulatory mechanisms, but the relationship

between miRNAs and CHL1 is unknown.

In our previous study, we found that miR-10a affects metastasis of colon cancer cells

(unpublished data). Thus, we presumed that miR-10a may regulate cell migration and/or

invasion in other cancer cells. This study focused on gaining a better understanding of

miR-10a expression at the molecular and cellular levels in HeLa and C33A human

cervical cancer cell lines. We demonstrated that overexpression of miR-10a promoted

colony formation, migration and invasion as well as reduced CHL1 mRNA and protein

levels in cervical cancer cells. Illumination of the molecular mechanisms of miR-10a in

the initiation and progression of human cervical cancer could provide a treatment strategy

for cervical cancer.

2. Materials and methods

2.1. Vector construction

To construct a miR-10a expressing vector (pcDNA3/pri-miR-10a, pri-miR-10a), we

first amplified a DNA fragment carrying pri-miR-10a from genomic DNA using the

primers Pri-10a-sense and -antisense. The amplified fragment was then cloned into the

pcDNA3 at the HindIII and XbaI sites.

To construct the EGFP-CHL1 3’UTR reporter vector pcDNA3/EGFP-CHL1-3’UTR, a

234 bp fragment of CHL1 3’UTR containing the predicted miR-10a binding site was

amplified by RT-PCR using the primers CHL1-3’UTR-sense and -antisense. The PCR

product was inserted into the vector backbone downstream of the EGFP gene between the

BamHI and EcoRI sites of the pcDNA3/EGFP vector as previously described [33]. In

addition, the 3’UTR fragment containing the mutated miR-10a binding site was amplified

using PCR side-directed mutagenesis assay. The two additional primers were CHL1-

3’UTR-MS and -MA. The PCR product was insert into pcDNA3/EGFP with the same

sites to form the reporter vector pcDNA3/EGFP-CHL1-3’UTRmut.

The pSilencer/shRNA-CHL1 plasmid expressing a small interfering RNA (siRNA)

targeting the CHL1 transcript was constructed by annealing a single-strand hairpin DNA

and inserting it into a pSilencer2.1/neo vector (Ambion) using the BamHI and HindIII

sites.

To construct the CHL1 expression plasmid, the coding sequence (open reading frame,

ORF) without the 3’UTR of human CHL1 (NM_006614) was purchased from Sino

Biological Inc. The 3699-bp fragment was subcloned into the EcoRI and XhoI sites of

pcDNA3. All of the primers used are shown in Table 1.

2.2. Cell culture and transfection

HeLa and C33A cells were grown in RPMI 1640 (GIBCO) supplemented with 10%

and 20% fetal bovine serum, respectively, along with 100 IU/ml of penicillin and 100

μg/ml of streptomycin. The cells were incubated at 37°C in a humidified chamber with

5% CO 2 . The transfection was performed with the Lipofectamine 2000 Reagent

(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.

2.3. MTT assay

HeLa cells and C33A cells, following transfection, were seeded in 96-well plates at a

density of 8000 cells per well. Then, 24 h, 48 h, 72 h after transient transfection, the cells

were incubated with 10 µl MTT (at a final concentration of 0.5 mg/ml) at 37°C for

another 4 h. The medium was then removed, and the precipitated Formosan was

dissolved in 100 µl DMSO. After shaking for 20 min, the absorbance at 570 nm (A 570 )

was detected using an uQuant Universal Microplate Spectrophotometer (Bio-Tek

Instruments, Winooski. USA).

2.4. Colony formation assay

Following transfection, the HeLa cells and C33A cells were counted and seeded in 12-

well plates (in triplicate) at a density of 100 cells per well. The plates were incubated at

37°C and 5% CO 2 in a humidified incubator. Fresh culture medium was replaced every

3 days. After 10 days of culture, the cells were stained with crystal violet, and the

numbers of colonies containing more than 50 cells were counted. The rate of colony

formation was calculated using the following equation: colony formation rate = (number

of colonies/number of seeded cells) × 100%

2.5. In vitro migration and invasion assays

For the transwell migration or invasion assays, HeLa or C33A cells in 0.2 ml RPMI

1640 without fetal bovine serum (FBS) were placed on the top chamber of each insert

(BD Biosciences, NJ) with or without 40 μl of 1 mg/ml Matrigel. The lower chamber was

filled with 600 µl of RPMI 1640 medium with 10% FBS to act as the nutritional

attractant. Twenty-eight hours later, the migrant cells that had attached to the lower

surface were fixed with 20% methanol and stained for 20 min with crystal violet. The

membranes were then carved and embedded under cover slips with the cells on the top.

Cells in three different fields of view at 100 × magnification were counted and expressed

as the average number of cells per field of view. All assays were performed in triplicate.

2.6. miRNA target prediction

The putative targets of miRNA were predicted using the PicTar, TargetScan and

miRBase Targets [34] databases.

2.7. Real-time RT-PCR

Real-time RT-PCR was performed to detect the relative level of CHL1 transcription

and mature miRNA [35]. Briefly, complement DNA (cDNA) was generated through

reverse transcription using M-MLV reverse transcriptase (Promega, Madison, WI) with 5

µg of total RNA. This cDNA product was used to amplify the CHL1 gene and the β-actin

gene. The PCR conditions were as follows: 95°C for 3 min, followed by 40 cycles of

95°C for 30 s, 56°C for 30 s and 72°C for 30 s.

To detect the levels of mature miRNA, 5 µg of total RNA was reverse transcribed to

cDNA with specific RT primer, and stem-loop real-time PCR was used to detect miR-10a

level. The PCR cycles were as follows: 95°C for 3 min, followed by 40 cycles of 95°C

for 30 s, 56°C for 30 s and 72°C for 30 s. Real-time PCR was performed with the SYBR

Premix Ex Taq (TaKaRa, Otsu, Shiga, Japan) on an iQ5 Real-Time PCR Detection

System (Bio-Rad).

2.8. Western blot

The cells were lysed with RIPA lysis buffer, and the proteins were harvested. All

proteins were resolved on a 10% SDS denatured polyacrylamide gel and then transferred

onto a nitrocellulose membrane. The membranes were incubated overnight at 4°C with

polyclonal rabbit anti-human CHL1 (Saberio, China) and rabbit anti-human GAPDH

(Saierbio, China). The membranes were then washed and incubated with a horseradish

peroxidase-conjugated secondary antibody. Protein expression was assessed by enhanced

chemiluminescence and exposure to chemiluminescent film. Lab Works TM Image

acquisition and analysis software (UVP, Upland, CA, USA) was used to quantify the

band intensities.

2.9. Fluorescent reporter assay

For the fluorescent reporter assay, HeLa cells and C33A were seeded in a 48-well plate

the day before transfection. The cells were co-transfected with 0.4 µg of pcDNA3/pri-

miR-10a or control vector and 0.2 µg of CHL1-3’UTR or CHL1-3’UTRmut, with

pcDNA3/EGFP as a control. The red fluorescence protein (RFP) expression vector

pDsRed2-N1 (Clontech, Mountain View, CA, USA) was used for normalization. The

cells were lysed 72 h later, and the intensity of EGFP and RFP fluorescence was detected

with the F-4500 Fluorescence Spectrophotometer (Hitachi, Tokyo, Japan).

2.10. Clinical cervical cancer specimens and RNA isolation

Paired samples of human cervical cancer tissue and corresponding adjacent cervical

tissue were obtained from the Cancer Center of Sun Yat-sen University of Medical

Sciences. All of the samples were obtained with the patients’ informed consent and

approved by the Ethics Committee of Sun Yat-sen University of Medical Sciences. The

category of the specimens was confirmed by pathological analysis. Large and small

RNAs were isolated from tissue samples using the mirVana™ miRNA Isolation Kit

(Ambion, Austin, TX, USA), according to the manufacturer’s instructions.

2.11. Statistical analysis

The hypothesis test for significance between two groups with complete randomized

design or paired design utilized the Student’s t test, and the test for significance between

three groups utilized one-way analysis of variance (ANOVA) followed by Student-

Newman-Keuls q test to compare two groups. The statistical significance was set to P <

0.05.

3. Results

3.1. MiR-10a promotes the growth of human cervical cancer cells

To determine whether miR-10a affected cell viability and growth, cervical cancer

HeLa cells were transfected with pcDNA3/pri-miR-10a. The mature miR-10a level was

significantly increased in pri-miR-10a transfected HeLa (nearly one-fold) or C33A (more

than one-fold) cells. And it was significantly reduced in 2'-O-methyl-modified antisense

oligonucleotide of miR-10a (ASO-miR-10a) transfected HeLa (nearly 80%) or C33A

(nearly 90%) cells (Fig. 1A). Based this observation, we transfected HeLa cells with

either pcDNA3/pri-miR-10a or ASO-miR-10a and performed the MTT assay to measure

cell viability. This result indicated that pri-miR-10a and ASO-miR-10a did not

significantly influence HeLa cell viability at 24 h, 48 h and 72 h after transfection,

compared with the control vector or the negative control of antisense oligonucleotide

(ASO-NC) oligomer (Fig. 1B). Similar results were found in the C33A cell line (Fig. 1C).

However, the colony formation rate in HeLa cells transfected with pcDNA3/pri-miR-10a

increased by approximately 50% more than that in control vector transfected cells (Fig.

1D). Inversely, ASO-miR-10a transfection decreased the colony formation rate in HeLa

cells by approximately 50% (Fig. 1E). Similar data were available in C33A cells (Fig. 1F

and G). These results demonstrated that alteration of the miR-10a levels does not affect

cell viability over a short duration but does regulate the capacity of cell proliferation in

cervical cancer cell lines.

3.2. MiR-10a promotes the migration and invasion of cervical cancer cells in vitro

It has been previously reported that CHL1 plays an important role in cell migration and

invasion. Therefore, we investigated whether targeting of miR-10a to CHL1 was

associated with the migration and invasion properties of cervical cancer cells. Transwell

assays without Matrigel were used to detect the effects of miR-10a on the migratory

potential of cells, and transwell assays with Matrigel were used to demonstrate the effects

of miR-10a on the invasive potential of cells. The ectopic expression of miR-10a resulted

in an approximately one-fold increase in HeLa cell migration (Fig. 2A). To determine

whether the loss of miR-10a would inhibit the migration of HeLa cells, we silenced miR-

10a with ASO-miR-10a in the HeLa cells, which resulted in a almost 60% reduction in

cell migration (Fig. 2B). Similarly, miR-10a could promote the invasive activity in the

two cell lines (Fig. 2C and D). These data indicate that miR-10a promotes the migration

and invasion of cervical cancer cells in vitro.

3.3. MiR-10a targets CHL1 and negatively regulates its expression

MiRNAs can regulate mRNA stability and the translation of target mRNA at the

post-transcriptional level [36]. To determine whether miR-10a regulates the expression of

CHL1, we investigated the endogenous mRNA and protein expression levels of CHL1

through quantitative RT-PCR analysis and Western blotting. The CHL1 mRNA level was

significantly reduced by nearly 65% in HeLa cells transfected with pcDNA3/pri-miR-10a

but significantly increased by approximately 50% in HeLa cells transfected with ASO-

miR-10a (Fig. 3A). Furthermore, the overexpression of miR-10a led to an approximate

30% reduction of the CHL1 protein level, but knockdown of miR-10a through ASO-miR-

10a resulted in a nearly one-fold increase of the CHL1 protein level (Fig. 3B). Similar

results were observed in C33A cells (Fig. 3A and B). These data suggested that miR-10a

negatively regulates the endogenous CHL1 protein expression through both mRNA

degradation and translational repression.

To detect the expression of miR-10a in human cervical cancer tissue, we investigated

the relationship between miR-10a and CHL1 in 14 pairs of cervical cancer and adjacent

normal tissues by real-time RT-PCR assay. After normalization to the U6 control, miR-

10a was expressed at significantly higher level in tumor tissues compared with the

corresponding non-tumor tissues (Fig. 3C). After normalization to the β-actin control,

CHL1 was expressed at significantly lower levels in the tumor tissues compared with the

corresponding non-tumor tissues (Fig. 3C). Thus, we can speculate that miR-10a might

negatively regulate the expression of CHL1.

MiRNAs regulate a variety of cellular activities and biological functions by regulating

the expression of target genes. To investigate the relationship between miRNAs and

CHL1, three algorithm programs (PicTar, TargetScan Release 5.2 and miRBase Targets)

were used to predict the miRNA targets in the 3’UTR of CHL1. A putative binding site

for miR-10a was found in the 3’UTR of CHL1 (Fig. 3D). To test if miR-10a was able to

directly bind to the CHL1 3’UTR and repress CHL1, we constructed an EGFP reporter

vector (pcDNA3/EGFP-CHL1-3’UTR) with the miR-10a binding sequence of the

putative CHL1 3’UTR placed downstream of the EGFP stop codon. HeLa cells were

cotransfected with pcDNA3/pri-miR-10a, ASO-miR-10a oligonucleotides, or control

vector. The intensity of the EGFP fluorescence in cells transfected with pcDNA3/pri-

miR-10a was significantly reduced nearly 40% after 48 h compared to the control cells,

and the intensity of EGFP fluorescence in cells transfected with ASO-miR-10a was

significantly increased by almost 45% compared to the control cells (Fig. 3E).

Furthermore, we constructed an additional EGFP reporter vector containing the CHL1

3’UTR with a mutant miR-10a binding site to determine the function of the miR-10a

binding site (Fig. 3D). As a result, pcDNA3/pri-miR-10a and ASO-miR-10a had no

effect on the intensity of EGFP fluorescence in cells transfected with the 3’UTR mutant

vectors (Fig. 3E). Similar results were observed in C33A cells (Fig. 3F).

3.4. CHL1 inhibits cell colony formation, migration and invasion in vitro

To test whether CHL1-regulated proliferation, migration and invasion of cervical

cancer cells was mediated by miR-10a, we used a pSilencer/sh-CHL1 plasmid to

knockdown CHL1 by small hairpin RNA (shRNA) expression. To test whether

pSilencer/sh-CHL1 plasmid can lead to CHL1 knockdown, we investigated the mRNA

and protein expression levels of CHL1 through quantitative RT-PCR analysis and

Western blotting in cervical cancer cells transfected with either the pSilencer/sh-CHL1

plasmid or pSilencer vector. The CHL1 mRNA level was significantly reduced almost

80% in pSilencer/sh-CHL1 transfected HeLa and nearly 40% in C33A cells (Fig. 4A).

Consistent with these results, the protein expression level of CHL1 was significantly

reduced almost 50% in the pSilencer/sh-CHL1 transfected HeLa and nearly 25% in C33A

cells (Fig. 4B). However, CHL1 knockdown by pSilencer/sh-CHL1 did not affect cell

viability of HeLa and C33A cells (Fig. 4C and D), but it resulted in an approximately

50% increase of the colony formation rate (Fig. 4E and F). Moreover, CHL1 knockdown

resulted in an almost two- or one-fold increase in HeLa or C33A cell migration,

respectively. (Fig. 4G). Similar results were observed in the invasive assays (Fig. 4H).

Next, a pcDNA3/CHL1 vector (a CHL1 overexpression vector without the 3’UTR) was

constructed to reverse the effects of miR-10a. When HeLa cells were cotransfected with

pcDNA3/pri-miR-10a and pcDNA3/CHL1, the facilitation of colony formation,

migration and invasion caused by pcDNA3/pri-miR-10a was reversed by the expression

of pcDNA3/CHL1 compared to the control vector. First, we investigated the mRNA and

protein expression levels of CHL1 through quantitative RT-PCR analysis and Western

blotting. In HeLa and C33A cells cotransfected with pcDNA3/pri-miR-10a and

pcDNA3/CHL1, the CHL1 mRNA level was higher than both the pcDNA3/pri-miR-10a

with pcDNA3 group (60%) and the pcDNA3 group (20%) but lower than the pcDNA3

with pcDNA3/CHL1 group (25%) (Fig. 5A and B). In addition, the protein expression

level of CHL1 was higher than both the pcDNA3/pri-miR-10a with pcDNA3 group (one-

fold) and the pcDNA3 group (10%) but lower than the pcDNA3 with pcDNA3/CHL1

group (30%) (Fig. 5C and D). These results suggested that our rescue experiment was

effective. In addition, the overexpression of CHL1 using pcDNA3/CHL1 in cells

cotransfected with pcDNA3/pri-miR-10a led to a nearly 40% reduction of the colony

formation rate in HeLa and C33A cells compared to the pcDNA3/pri-miR-10a with

pcDNA3 group. In other words, the effect of miR-10a on cell growth of cervical cancer

cells was counteracted (Fig. 5E and F). Similarly, the group in which CHL1 and miR-10a

were both overexpressed resulted in an approximately 50% or 30% reduction in HeLa or

C33A cells migration (Fig. 6A), Similar results were available in the invasive assays (Fig.

6B). Together, these results provide further evidence that CHL1 is a tumor suppressor

gene. Therefore, ectopic expression of CHL1 might rescue the increase of growth,

migration and invasion caused by miR-10a. Because of the tumor suppressor role that

CHL1 plays in cervical cancer, we could explain why overexpression of miR-10a

promotes colony formation, migration and invasion of cervical cancer cells.

4. Discussion

Recently, increasing evidence has revealed the abnormal expression of miRNAs in

various types of tumors. Thus, more researchers focused on the tumor-associated

miRNAs and specific target genes to determine the mechanism of action [37]. The

identification of cancer-specific miRNAs and their targets is critical for understanding

their role in tumor invasion and metastasis and might be essential for defining novel

therapeutic targets [38-40]. The members of the miR-10 family have been reported to be

deregulated in several cancer forms [41]. Both miR-10a and miR-10b are overexpressed

in primary hepatocellular carcinomas, when compared with the levels in normal liver

[42]. Furthermore, miR-10a was found to be overexpressed in colon cancer [43] and was

involved in pancreatic cancer invasion and metastasis [44,45]. In a controversial paper,

miR-10b was related to tumor invasion and metastasis in breast cancer [44,45].

Therefore, we wanted to determine whether miR-10a is deregulated in cervical cancer

and whether it has the ability to promote cervical cancer cell metastasis.

In this study, we detected the expression of miR-10a in 14 pairs of human cervical

cancers and normal tissues by Real-time PCR. The expression of miR-10a was found to

be increased in 13 pairs of the human cervical cancer tissues, which is consistent with

previous reports [42,43]. The upregulation of miR-10a in human cervical cancer may

contribute to the initiation and progression of tumors, which suggests that miR-10a may

play an oncogenic role. Prior to this study, however, the role of miR-10a and its target

genes was unclear in cervical cancer. Therefore, the functions and molecular mechanisms

of miR-10a in human cervical cancer were the focal point of our research.

To characterize miR-10a action in human cervical cancer, we detected the changes in

cellular phenotypes after overexpression of the primary transcript of miR-10a and the

inhibition miR-10a. We performed the MTT assay to measure cell viability in HeLa and

C33A cells and found that miR-10a did not affect cell viability (Fig. 1B and C). In the

colony formation assay, however, we found that pri-miR-10a promotes the colony

formation activity of HeLa and C33A cells (Fig. 1D-G). These results demonstrated that

the ectopic expression of miR-10a does not affect cell viability within a short time period

but enhanced the proliferation of cervical cancer cell lines. Transwell assays with or

without Matrigel were performed to detect the functions of miR-10a in HeLa cell

migration and invasion. Our results demonstrated that overexpression of pri-miR-10a

significantly promoted the migration and invasion of HeLa and C33A cells compared to

the control. Conversely, when transfected with ASO-miR-10a, the migratory and invasive

capacities were dramatically reduced in HeLa and C33A cells. In summary, miR-10a

promotes cervical cancer colony formation, migration and invasion in HeLa cell and

C33A cells.

In this study, we focused on the role of miR-10a in targeting CHL1 in human HeLa

cells. Using biological information technology, we searched for target genes of miR-10a,

and we were interested in a CHL1. CHL1 is a type I transmembrane protein that belongs

to the L1CAMs, which are expressed during neuronal development and are essential in

the nervous system [46,47]. As reported previously [48,49], primary tumor tissues and

various human cancer cell lines can express CHL1. These results suggest that CHL1

could be involved in development of cancer. Because miR-10a is highly expressed in

cervical cancer and is associated with malignant phenotypes, miR-10a may target genes

with anti-proliferative activity and anti-malignant transformation. Moreover, we found a

lower expression level of CHL1 in cervical cancers compared to adjacent normal tissues

and an inverse correlation between the expression of miR-10a and CHL1 in tumor

tissues. Furthermore, the EGFP fluorescence intensity of EGFP-CHL1-3’UTR was

specifically responsive to miR-10a overexpression. Mutation of the miR-10a binding site

abolished the miR-10a effect on the regulation of EGFP fluorescence intensity (Fig. 3E

and F). Finally, we found that both CHL1 mRNA and protein levels were decreased in

HeLa and C33A cells transfected with pri-miR-10a but were increased in HeLa and

C33A cells transfected with ASO-miR-10a (Fig. 3A and B). These results suggested that

CHL1 was a target of miR-10a and was negatively regulated, and it may have anti-

proliferative activity and anti-malignant transformation in the HeLa and C33A cells.

Various recent studies have suggested that CHL1 is most frequently overexpressed in

major human cancers and functions as an oncogene to promote metastasis [26]. However,

our findings demonstrated that CHL1 is poorly expressed in cervical cancer tissue

specimens, HeLa cells and C33A cells compared to the non-tumor tissue specimens,

which is entirely different from the reported expression pattern of CHL1 in breast and

ovarian cancer. Because we observed different results in different cancer types, we

further assessed whether miR-10a promotes the colony formation, migration and invasion

of HeLa and C33A cells by regulating CHL1. We found that knockdown CHL1 could

enhance colony formation, cell migration and invasion capacity (Fig. 4). Moreover,

ectopic expression of CHL1 without the 3’UTR rescued the ability of miR-10a to

promote colony formation, cell migration and invasion in HeLa cells and C33A (Figs. 5

and 6). Together, these results demonstrated that miR-10a reduces CHL1 expression

levels and promotes colony formation, cell migration and invasion of cells.

In our study, we found that CHL1 shows anti-proliferative activity and anti-malignant

transformation in the HeLa and C33A cells, which is contrary to previous reports. This

may be due to the malignant state, type, and stage of the cancer. In different tumors and

stages, CHL1 may play dual roles. It was reported that CHL1 negatively regulates the

proliferation and neuronal differentiation of neural progenitor cells through activation of

the MAPK pathway [50]. And CHL1 cooperates with PAK1-3 to regulate morphological

differentiation of embryonic cortical neurons [46]. PAK family kinases was known to be

closely related with tumor migration and invasion [51,52], so we presume that in cervical

cancer cells, low expression of CHL1 leads to dysregulation of the MAPK and PAK

pathways, and then affects the downstream molecules, finally contributes cell growth,

migration and invasion in human cervical cancer cells (Fig. 7). In addition, CHL1 is

located at 3p26 and is involved in some neurological diseases [53]. However, the

function of CHL1 on tumor progression was less studied, and the mechanism of CHL1 in

tumor progression is a key question in biology. Therefore, we expect more reports on

CHL1.

In summary, we demonstrated that miR-10a expression is up-regulated in cervical

cancer tissues, and miR-10a promotes cell growth, migration and invasion by targeting

CHL1 in human cervical cancer cells. These results suggest that miR-10a could have an

important role in tumor metastasis by regulating CHL1.

Many other mRNAs display different expression patterns in variety of malignancies

and play similar mechanisms in tumor development and other biological processes

[54,55]. In future research, we look forward to the discovery of additional tumor-

associated miRNAs and their roles in the downstream regulatory pathways. The

relationship between miRNAs and the specific mechanism of tumor formation will

become clearer. Furthermore, new insights into cancer treatment and prevention will be

offered.

5. Conflict of interest

None declared.

Acknowledgements

We thank the Cancer Center of Sun Yat-sen University of Medical Sciences for providing

the cervical cancer tissues. We also thank the College of Public Health of Tianjin

Medical University for the technical assistance in fluorescence detection. This work was

supported by the National Foundation of China (No. 30873017, 31071191, 91029714).

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Figure legends

Fig. 1. High expression levels of miR-10a promote the proliferation of HeLa and C33A

cells in vitro. (A) HeLa and C33A cells were transfected with pcDNA3/pri-miR-10a,

ASO-miR-10a or the negative controls, and miR-10a was detected by quantitative RT-

PCR using U6 snRNA for normalization. (B) HeLa cells were transfected with either

pcDNA3/pri-miR-10a or ASO-miR-10a. The HeLa cell viability was detected by the

MTT assay at 24 h, 48 h and 72 h after transfection. (C) Similar results as in part B were

obtained in C33A cells. (D, E) HeLa cells were transfected with either pcDNA3/pri-miR-

10a (D) or ASO-miR-10a (E) in 48-well plates and then seeded in 12-well plates. The

number of colonies was counted on the 2 weeks after seeding, and finally the cells were

stained with crystal violet The representative images of colonies are shown. (F, G) C33A

cells were transfected with either pcDNA3/Pri-miR-10a (F) or ASO-miR-10a (G). The

results were similar to the colony formation results in HeLa cells. The data are presented

as the means ± SD of three independent experiments (*P < 0.05, **P < 0.005).

Fig. 2. MiR-10a promotes cell migration and invasion of HeLa and C33A cells in vitro.

(A ， B) Transwell migration assays were carried out with HeLa and C33A cells

transfected with either pcDNA3/pri-miR-10a (A) or ASO-miR-10a (B). Representative

fields of migratory cells on the membrane were presented below. The histogram shows

the relative number of migratory cells from three independent experiments. (C, D)

Overexpression (C) and knockdown (D) of miR-10a in HeLa and C33A cells showed

similar results in the invasive assays as in the migration assays (*P < 0.05, **P < 0.005).

Fig. 3. Direct regulation of CHL1 by miR-10a through binding to the 3’UTR of CHL1

mRNA. (A, B) The mRNA (A) or protein (B) levels of CHL1 were tested by quantitative

RT-PCR or Western blotting, respectively. Normalization was performed with β-actin or

GAPDH. (C) The relative expression level of mature miR-10a in the 14 cervical tissue

pairs, including cancer tissues (Tumor) and matched normal tissues (Normal), was

detected by quantitative RT-PCR using the U6 snRNA for normalization. CHL1 mRNA

expression level was detected by quantitative RT-PCR using β-actin for normalization.

The data represent the means ± SD of three independent experiments. (D) Sequence

alignment of miR-10a (mid) with the wild-type (top) and mutated (bottom) 3’UTR of

CHL1. The arrows indicate the mutational nucleotides. (E, F) The fluorescent reporter

vectors were co-transfected with pcDNA3/pri-miR-10a or ASO-miR-10a into HeLa (E)

and C33A (F) cells, and fluorescence intensity of the protein extract was detected. The

means of EGFP fluorescence intensity were normalized by RFP. For all panels, one

representative experiment is shown out of at least three experiments (*P < 0.05, **P <

0.005).

Fig. 4. CHL1 knockdown promotes colony formation, migration and invasion in HeLa

and C33A cells. (A) The relative expression level of CHL1 mRNA in HeLa cells

transfected with pSilencer/sh-CHL1 vector was detected by quantitative RT-PCR using

β-actin for normalization. (B) Western blotting analysis showed that CHL1 knockdown

was effective. (C, D) The MTT assay indicated that CHL1 knockdown did not influence

HeLa and C33A cells viability. (E, F) The colony formation assay revealed that the

average colony formation rate was increased by CHL1 knockdown. (G, H) The average

number of migratory (G) and invasive (H) cells from three independent experiments ±

SEM. Representative fields of migratory and invasive cells on the membrane were

presented below (*P < 0.05, **P < 0.005).

Fig. 5. CHL1 alleviates miR-10a-induced colony formation capacity in vitro. (A, B) The

relative expression level of the CHL1 mRNA without a 3’UTR was detected by

quantitative RT-PCR using β-actin for normalization in HeLa (A) and C33A (B) cells.

(C, D) Western blotting analysis showed that ectopic expression of CHL1 without 3’UTR

rescued the decreased CHL1 protein caused by pri-miR-10a. (E, F) The colony formation

assay revealed that with present of miR-10a, the average colony formation rate was

decreased by the expression of CHL1 without a 3’UTR.

Fig. 6. CHL1 alleviates miR-10a-induced migration and invasion capacity in vitro. (A)

Ectopic expression of CHL1 without a 3’UTR decreased HeLa and C33A cells migration

with present of miR-10a. The average number of migratory cells was from three

independent experiments ± SD. (B) Similar results were available in C33A and HeLa

cells for the invasive activities. (*P < 0.05, **P < 0.005).

Fig. 7. Possible regulation pathways of CHL1 in cervical cancer cells. Overexpression of

miR-10a in cervical cancer cells leads to low expression of its target CHL1 and then

affects the PAK1-3 and MAPK pathway, finally contributes to the malignant phenotypes

of the cancer cells.

Table 1. Sequence of the primers used in this study.

Primer Sequence (5’ to 3’)

Pri-10a-sense GACTGAAGCTTAATCCCAAGAACAGACTCGCAC

Pri-10a-antisense CCACGTCTAGACGTGGGGAGAGTTCAGGTAGATG

ASO-miR-10a cacaaauucggaucuacagggua

ASO-NC gacuacacaaaucagcgauuu

miR-10a-RT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACAAATTC

U6-RT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATATGGAAC

miR-10a-Fwd TGCGGTACCCTGTAGATCCG

U6-Fwd TGCGGGTGCTCGCTTCGGCAGC

Reverse primer CCAGTGCAGGGTCCGAGGT

CHL1-siR-Top GATCCGGCTATCAGATAAATTGGTTTCAAGAGAACCAATTTATCTGATAGCCTTTTTTGGAAGAATTCA

CHL1-siR-Bot AGCTTGAATTCTTCCAAAAAAGGCTATCAGATAAATTGGTTCTCTTGAAACCAATTTATCTGATAGCCG

CHL1-3'UTR-sense CGTGGATCCGTTCCAGCACTGATTTTGTAC

CHL1-3'UTR-antisense GCGGAATTCGGTGACTTTCAGATACATAATTAG

CHL1-3'UTR-mut-sense CCTTATCTTTGTCAGTCCGAGTTCTTTTTTATGAAG

CHL1-3'UTR-mut antisense

CTTCATAAAAAAGAACTCGGACTGACAAAGATAAGG

CHL1-sense CGGAATTCTCCACCATGGAGCCGCTTTTACTTG

CHL1-antisense GCTGTCTCGAGTATGCCCGAAGGGGAAAAG

β-actin-sense CGTGACATTAAGGAGAAGCTG

β-actin-antisense CTAGAAGCATTTGCGGTGGAC

类别：临床实验室生物安全管理及分子诊断技术与临床应用884338 肺腺癌早期诊断血清标志物microRNA 的筛选及鉴定

吕少刚，明亮（郑州大学第一附属医院检验科）

摘要目前，肺癌是世界范围内发病率最高的恶性肿瘤，也是癌症相关致死率最高的

恶性肿瘤，而肺腺癌是占肺癌中最多的一种组织学分型。现在还没有灵敏度和特异度都比较高的肺腺癌早期诊断标志物，导致大多数肺腺癌患者都是在晚期才确诊，因 此 急 需 筛 选 出 有 效 的 肺 腺 癌 早 期 诊 断 标 志 物 。 研 究 表 明 血 清microRNA（miRNA）可作为一种新型的肿瘤诊断标志物，因此我们对比肺腺癌患者与健康对照血清miRNA的表达差异情况，以筛选并鉴定潜在的肺腺癌早期诊断标志物。我们收集了 180名肺腺癌患者和 180名健康对照的血清样本，先将 20份肺腺

癌患者的血清和 20份健康对照的血清分别混合并提取 RNA，利用低密度基因芯片（low-density array，LDA）分别检测两者的miRNA表达谱，并统计其差异表达的miRNA。选择差异表达最明显的一组 miRNA，并用茎环法实时定量 PCR（real

time quantitative PCR，RT-qPCR）验证剩余的 160份肺腺癌和 160份健康对照的血清。结果发现，混合样本中有 11个miRNA在肺腺癌患者血清中表达明显高于健康对照，从中选取 6个miRNA，包括miR-103、miR-146a、miR-151、miR-21、miR-

221、miR-222和miR-223，用 RT-qPCR进行鉴定。最后发现表达差异最大的三个miRNA，miR-146a、miR-222和miR-223，在 I/II期肺腺癌患者血清中表达水平明显高于健康对照。综上所述，miR-146a、miR-222和miR-223组成的 miRNA 组可以作为肺腺癌

早期诊断的潜在标志物。

关键词：肺腺癌，早期诊断，microRNA

1. 前言肺癌是目前世界范围内患病率最高的恶性肿瘤，也是肿瘤相关致死的首要原因

[1-3] 。临床上，肺癌主要分为两大类：小细胞 肺癌（ small cell lung

cancer，SCLC）和非小细胞肺癌（non-small cell lung cancer，NSCLC），NSCLC

占所有肺癌的 85% ~90%[4]。NSCLC 在组织学分类上又分为四种：腺癌（ adenocarcinoma，AD）、鳞癌（ squamous cell carcinoma，SQ）、大细胞癌（large cell carcinoma，LC）和其他。其中，腺癌是 NSCLC中最常见的组织类型，世界范围内每年约有 500, 000病例因其致死[5]。在中国和其他东亚地区人群中，尤其是女性和非吸烟者，肺腺癌患病率明显较高，然而肺腺癌患者的五年生存率只有约 15% [6, 7]。肺腺癌患者的生存和预后与首次确诊时的肿瘤分期紧密相关，但是由于目前缺

乏有效的早期诊断方法，导致大多数肺腺癌患者直到晚期才被确诊，使得肺腺癌病死率非常高。目前临床上仍以影像学和细胞生物学为基础的筛查方式作为肿瘤早期诊断，研究证明其对某些肿瘤具有较高的灵敏度，但是对肺腺癌早期诊断和降低肺腺癌的病死率并没有表现出明显的效果[8, 9]。因此，目前急需发展可用于肺腺癌早期筛查的高灵敏度标志物。

miRNA是一类小的非编码 RNA，长度约为 22个核苷酸，通过与 mRNA的 3’

UTR结合，降解 mRNA或者抑制其翻译，从而达到在转录后水平负性调节基因表达的效果。miRNA 通过转录后水平调节机制可以影响很多种细胞功能，包括细胞生长、细胞分化以及细胞凋亡等，研究发现 miRNA在肿瘤生成中也起着重要的作用[10-12]。越来越多的证据表明，miRNA在肿瘤的发生发展、肿瘤的迁徙、肿瘤的侵袭以及肿瘤的转移过程中均起着非常重要的作用[13-17]。近年来，有研究发现miRNA在血清中可以稳定存在，并且血清 miRNA的表达水平与很多种肿瘤的发生发展以及预后都有着密切的关系，比如肺癌、结直肠癌、前列腺癌、胃癌以及骨肉

瘤等[18-24]。因此，血清 miRNA具有成为新型的肿瘤早期诊断及预后标志物，其有以下两大优点：（i）血清miRNA可以直接用 RT-qPCR检测，该技术在临床实验室应用广泛；（ii）用血液中的肿瘤标志物筛查高危人群和肿瘤的早期诊断可以减少对受试者造成的损伤，属于非侵入性检查。目前已经有相关研究评价血清 miRNA作为 NSCLC的早期诊断标志物的潜力。

Roth等人的研究报告称血清 miR-361-3p和miR-625作为标志物可以有效的鉴别肺癌和良性肿瘤 [25]。另外， Shen 等人的研究表明， miRNA-21、 miRNA-

126、miRNA-210和 miRNA-486-5p等四个 miRNA在诊断 NSCLC时，灵敏度为86.22%，特异度为 96.55%，因此极具潜力成为 NSCLC的标志物[26]。近年来 Dou

等学者发现相对于健康对照，NSCLC患者血浆内 let-7c和 miR-152明显降低， ROC曲线下面积 AUC分别为 0.714和 0.845，表明这两个miRNA具有成为NSCLC

筛查及诊断标志物的潜力[21]。然而，肺腺癌作为 NSCLC的最主要分型，其血清 miRNA表达谱到目前为止

还不甚明了。因此，为了发展以 miRNA为标志物进行鉴别肺腺癌患者和健康人这种非侵入性检测方法，我们用低密度基因芯片和 RT-qPCR的方法评估了 180个肺腺癌患者和 180个健康对照的血清 miRNA表达谱，然后根据国际肺癌研究协会（international association for the study of lung cancer，IASLC）的肺癌分期体系，用AUC评估了差异表达的miRNA对不同时期肺腺癌的诊断能力。

2. 材料和方法2.1血清样本所有的样本均采集自上海市胸科医院，根据上海市胸科医院伦理委员会所要求

的流程，经伦理委员会批准同意，所有血液供体都签署了知情同意书。在此课题研究中，一共收集到 180例在上海市胸科医院首次确诊为肺腺癌并开始接受诊疗的患者血清，和 180例健康对照的血清。为了鉴定可能作为肺腺癌标志物的 miRNA，我们设计了一个多阶段病例对照研究。首先，分别将 20例肺腺癌患者血清和 20例

健康对照的血清混合组成两个样本池，通过高通量 PCR检测 766个miRNA的表达谱，从而筛选出肺腺癌患者和健康对照之间表达差异的血清 miRNA。然后，利用茎环法 RT-qPCR 分别检测剩余 160 例肺腺癌患者和 160 例健康对照的血清miRNA，从而验证第一步中筛选出来的差异表达的血清 miRNA，这一部分又分为两步：第一步，标志物选择，即先分别检测 40例肺腺癌患者和 40例健康对照的血清miRNA，根据一定的标准初步选择出一批 miRNA作为待选miRNA；第二步，标志物验证，即分别检测剩余的 120例肺腺癌患者和 120例健康对照的血清待选miRNA，确认最终可作为肺腺癌血清标志物的 miRNA。所有的患者都是2014~2015年间确诊为肺腺癌的，并在患者接受手术、化疗及放疗等临床治疗之前采集血液样本。最终肺腺癌诊断由手术中取下的肿瘤组织经组织病理学确认，并根据 IASLC指南确定肿瘤分期，其中不适宜手术操作的肺腺癌患者由组织活检和影像学检查确定其组织病理学特征和肿瘤分期。患者和健康对照的人口统计及临床特征见表 1。健康对照组样本从上海市胸科医院的常规体检样本库中选择，健康对照组与肺腺癌患者组在年龄、性别和种族构成比上相匹配。

表 1. 肺腺癌患者和健康对照的人口统计及临床特征　 标志物选择阶段 标志物验证阶段 　 P值

AD 对照 P值 AD 对照 P值 AD

(n=40) (n=40) (AD vs. (n=120) (n=120) (AD vs. 选择阶段 vs.

变量 No. % No. % 对照) No. % No. % 对照) 验证阶段)

平均年龄（岁） 60.1±7.9 60.2±8.0 0.967 59.9±8.0 59.4±8.3 0.631 0.673

年龄（岁） ≤60 20 50% 21 52.5% 0.823 60 50% 60 50.0% 1.000 1.000

＞60 20 50% 19 47.5% 60 50% 60 50.0%

性别 女性 20 50% 20 47.5% 58 48.3% 59 49.2% 0.855

分化程度 差 3 7.5% 6 5% 0.251

中等 18 45% 59 49.2%

高 19 47.5% 46 38.3%

未知 0 0% ` 9 7.5%

肿瘤分期 Ⅰ 26 65% 72 60% 0.522

Ⅱ 10 25% 31 25.8%

Ⅲ 4 10% 10 8.3%

Ⅳ 0 0% 0 0%

　 未知 0 0% 　 　 　 7 5.8% 　 　 　 　

2.2样本处理和 RNA提取用来做高通量 PCR 分析的 20例肺腺癌患者和 20例年龄性别分布相似的健康

对照的混合血清样本：先从各样本中取等量（250 μL）血清混合均匀，分别成为病例和对照的样本库，利用 mirVana PARIS Kit 试剂 盒（ Ambion， Grand

Island，NY，USA）并参照其 RNA提取步骤提取两个样本库的 RNA，最后的RNA 沉淀块溶于 100 μL洗脱液并储存于-80 ℃冰箱备用。用来做 RT-qPCR分析的剩余 160例肺腺癌患者和 160例健康对照的血清样本：每例样本取 200 μL血清，用mirVana PARIS Kit试剂盒提取miRNA。2.3高通量 PCR分析利用 Exiqon高通量 PCR分析仪（Exiqon，丹麦）进行低密度基因芯片分析，

检测两个样本库（20例肺腺癌患者血清样本和 20例健康对照血清样本）中血清循环 RNA中的 766个miRNA的表达情况。2.4 RT-qPCR定量分析miRNA分 别 从 每 个 RNA 样 品 中 取 7 μL ， 根 据 PrimeScript RT Enzyme

System（TaKaRa，日本）的操作步骤，利用该试剂 盒进行反转录（ Reverse

Transcription，RT）得到 cDNA，并将反转录得到的 cDNA用不含 RNA酶的水按1:10 稀释。然后利用 SYBR Green I PCR Master Mix试剂盒（TaKaRa，日本），在罗氏 480实时定量 PCR仪（罗氏，瑞士）上进行 PCR反应。每个反应孔均为 20

μL反应体系，包含 2 μL cDNA，3 μL正向引物，1.4 μL 通用反向引物和 10 μL

SYBR Green I PCR Master Mix。热循环参数设置如为：95 ℃ 预变性 5min，95 ℃

变性 15s、60 ℃ 退火并延伸 1min共 40个循环。每个 PCR 反应循环结束后进行溶解曲线分析，以确定 PCR产物的特异性。用相应的合成 miRNA 寡核苷酸制作标准曲线，并根据标准曲线计算目标miRNA的绝对浓度。每个反应重复三次。2.5统计分析实验数据用 Stata数据分析系统（12.0 版，StataCorp）进行统计分析，数据用

中位数和四分位数间距表示。肿瘤患者和健康对照之间血清 miRNA表达量的差异

用非参数检验Mann-Whitney检验法进行比较，P值 < 0.05认为具有显著性统计学意义。每一个目标miRNA都建立了受试工作者特征曲线，并计算曲线下面积，评估检测方法对肺腺癌检测的灵敏度和特异度，并用 logistics回归分析法分析肺腺癌和血清miRNA表达水平之间的相关性。3.结果3.1肺腺癌患者血清miRNA表达谱检测我们最开始筛查 20例肺腺癌患者和 20健康人血清组成的两个血清样本池，用

Exqion 高通量 PCR分析仪进行低密度基因芯片分析，共检测了 766个miRNA的表达情况。对比肺腺癌患者和健康对照的血清miRNA表达谱，发现与健康对照相比，肺腺癌患者血清中的 miRNA大多数表达明显降低。当 miRNA表达水平在肺腺癌患者和健康对照之间至少相差 5 倍，并且 PCR过程中荧光值达到阈值时的循环数（Crossing Point，Cp值） ≤ 35时，才认为该 miRNA在肺腺癌患者和健康对照之间存在差异表达。根据上述判断标准，我们发现在这总的 766个miRNA中有 11个差异表达的miRNA可作为候选肺腺癌标志物（见图 1）。

图 1. 肺腺癌患者血清miRNA表达特征的鉴定利用高通量 PCR分析仪进行血清 miRNA表达特征的鉴定，当 miRNA表达水平在肺腺癌

患者和健康对照之间至少相差 5 倍，并且 Cp值 ≤ 35时，才认为该miRNA在肺腺癌患者和健康对照之间存在差异表达。3.2在大量肺腺癌患者中有 6个miRNA差异表达我们验证了在上一部分中筛选出的 11个差异表达的候选miRNA表达水平。我

们取 160例临床上病理诊断为肺腺癌的患者血清和 160例健康对照血清的 RNA，通过茎环法 RT-qPCR，确认上述 11个候选 miRNA在血清中的表达量。其中，肺腺癌患者和健康对照在年龄和性别上的分布相似，无显著差异（见表 1）。在检测血清 miRNA之前，我们先用人工合成的单链 miRNA校准品做 RT-

qPCR反应并制作了标准曲线，发现在 miRNA处于 1 fmol/L ~ 100 pmol/L 范围内时，其半对数图呈线性（见图 2），因此，我们认为用血清 miRNA做 RT-qPCR反应测

量其表达水平，其结果具有良好的可靠性和可重复性。重复孔之间 Cp值非常相似（ r2 = 0.990），表明 RNA 提取方法具有较好的再现性。在所有的 11 个候选miRNA中 has-miR-92a表现出非线性扩增，因此我们剔除了该miRNA。在miRNA标志物选择阶段，我们取 40例肺腺癌患者的血清 RNA和 40例健康

对照的血清 RNA分别做 RT-qPCR，选择在肺腺癌患者血清表达量平均高于健康对照的 1.5 倍，并且 P值 ≤ 0.005的miRNA进入后续验证实验。通过此标准，我们选出 6个在肺腺癌患者和健康对照之间差异表达的 miRNA，包括 miR-103，miR-

146a，miR-151-3p，miR-221，miR-222 和 miR-223（见表 2）。然后将这 6 个miRNA在 120例肺腺癌患者和 120例健康对照组成的更大批量样品中用 RT-qPCR

反应进一步验证，验证结果与前面的结果相同，这 6个miRNA在肺腺癌患者血清中的表达量明显高于健康对照，其升高范围为 3.46 – 11.59 倍（见表 2）。这 6个miRNA在标志物选择和标志物验证两个阶段共 160例肺腺癌患者和 160例健康对照血清样本中的表达差异情况见图 3。

表 2. 肺腺癌患者和健康对照血清中候选miRNA的浓度　 标志物选择阶段 　 标志物验证阶段

AD 对照 相差 AD 对照 相差　 (n=40) (n=40) 倍数 P值 　 (n=120) (n=120) 倍数 P值miR-103 17.20(27.70) 9.61(13.99) 1.79 <0.0005 17.15(27.46) 4.96(5.47) 3.46 <0.0001

miR-146a 35.20(46.20) 5.20(6.81) 6.77 <0.0001 53.00(47.30) 6.98(6.85) 7.60 <0.0001

miR-151-3p 3.86(3.22) 1.19(1.37) 3.25 <0.0001 3.29(3.14) 0.91(0.86) 3.62 <0.0001

miR-221 31.50(51.47) 12.56(12.56) 2.51 <0.0005 33.10(54.00) 6.76(11.28) 4.90 <0.0001

miR-222 14.30(14.29) 7.07(8.07) 2.02 <0.0003 21.70(19.80) 3.76(6.19) 5.78 <0.0001

miR-223 171.00(195.10) 31.80(53.60) 5.38 <0.0001 　 295.00(391.00) 25.45(38.20) 11.59 <0.0001

注释：miRNA 浓度单位是 fM/L，括弧前的数值表示 miRNA平均浓度，括弧内的数值表示四分位间距。

图 2. 用 RT-qPCR验证差异表达的miRNA

将人工合成的 miR-21、miR-23a、miR-103、miR-151-3p、miR-185、miR-223、miR-

146、miR-222、miR-221和miR-425分别按 10 倍的浓度差倍比稀释成 1 fM/L ~ 105 fM/L的系列浓度，根据 RT-qPCR结果制作标准曲线，横坐标为浓度（fM/L）的 log10对数值，纵坐标为 Cp

值。每次实验重复 3次，每个点的 Cp值用（均值±标准误）来表示。

图 3. 肺腺癌患者血清中表达上调的 6个miRNA

用 RT-qPCR方法检测 160例肺腺癌患者和 160例健康对照（包括所有标志物选择阶段和标志物验证阶段的样品）的 6个血清候选miRNA 浓度，根据标准曲线和 Cp值计算miRNA的绝对浓度，每个点表示一个样品三次重复检测的中位数。

图 4. 6个候选miRNA单独鉴别肺腺癌的 ROC曲线

6个候选miRNA单独鉴别肺腺癌和健康对照的能力，曲线下面积越大，表示独立鉴别肺腺癌的能力越大。3.3 3个miRNA适合作筛查肺腺癌的血清标志物为了进一步评估上述 6个候选miRNA作为肺腺癌血清标志物筛查肺腺癌的诊

断价值，我们评价了其诊断肺腺癌的灵敏度和特异度。根据 ROC曲线下面积选择了三个 AUC值最大的 miRNA（见图 4），miR-146a，miR-222和 miR-223组成miRNA 组，并建立 logistic回归模型，最佳预测公式为：Error: Reference source not found-2.06102+0.0508526×miR-146a-0.1463608×miR-

222+0.0226559×miR-223

根据以上公式，制作 miR-146a，miR-222和 miR-223所组成的 miRNA 组的ROC曲线并计算曲线下面积，发现 miRNA 组的 AUC值为 0.951（95%置信区间，0.92605 ~ 0.97644）（图 5A）。根据最佳 cutoff值（p = 0.5015），总的灵敏度（84.35%）和特异度（90.83%）达到该预测模型的最大值。分析结果确认了 miR-

146a，miR-222和miR-223所组成的 miRNA 组在检测肺腺癌中的效力，准确度高达 87.27%。

图 5. 根据 logistics回归分析绘制的miR-146a、miR-222和miR-223所组成miRNA 组的 ROC曲线

（A）miRNA 组鉴别肺腺癌和健康对照的 ROC曲线，（B）miRNA 组鉴别 IASLC 指南 TNM

分期 I期肺腺癌和健康对照的 ROC曲线，（C）miRNA 组 鉴别 IASLC指南 TNM分期 II期肺腺癌和健康对照的 ROC曲线，（D）miRNA 组 鉴别 IASLC指南 TNM分期 III期肺腺癌和健

康对照的 ROC曲线。3.4 3个miRNA与肺腺癌患者的肿瘤分期有关根据国际肺癌研究协会的肺癌 TNM分期指南，我们分析了血清 miRNA和肺

腺癌分期的关系（图 5B-D）。分析结果显示，在肺癌分期为 I、II和 III时，相应的 ROC曲线下面积分别是 0.942、0.968和 0.954，表明即使在肺腺癌早期（I期），miR-146a、miR-222和miR-223所组成的miRNA 组也能有效地检测出肺腺癌患者。3.5 3个miRNA对肺腺癌具有较高的特异度最后，我们利用鳞癌（ Squamous Cell Carcinoma， SQ）、肺炎（ Lung

Inflammation，INF）和小细胞肺癌（Small Cell Lung Cancer，SCLC）作为其他肺部疾病的对照，评估了 miR-146a、miR-222和miR-223所组成的miRNA 组对肺腺

癌的特异度。结果发现，现对于其他肺部疾病，在肺腺癌患者血清中 miRNA 组的表达水平明显升高（见图 6）。

图 6. miRNA 组 检测肺腺癌的特异度分析AD vs.control (P<0.001) ， AD vs.SQ (P<0.001) ， AD vs.INF (P<0.05) ， AD vs.SCLC

(P<0.01)。Y 轴表示 3 个 miRNA 的总浓度。* 表示 P<0.05，** 表示 P<0.01，*** 表示 P<0.001。4. 讨论目前，已有研究证明 miRNA可以稳定存在于血浆和血清中，并且在不同的肿

瘤，如肺癌、结直肠癌、前列腺癌及胰腺癌等患者血清中具有不同的表达谱[18,

19, 27]，这些发现表明循环 miRNA具有成为新型非侵入性肿瘤筛选标志物的潜力。然而，肺腺癌作为肺癌最主要的分型，在中国和其他东亚地区人群中，尤其是在女性和非吸烟者中，具有很高的发病率[6, 7]，但是到目前为止还没有发现肺腺癌的特异性肿瘤标志物，也没有发明出能有效检出早期肺腺癌的检测方法，因此急需鉴定出一种可以检测早期肺腺癌的高灵敏度肿瘤标志物。所以，我们旨在鉴定肺腺癌血清miRNA表达谱，以发现肺腺癌早期诊断标志物。在此次研究中，为了检测中国的肺腺癌患者血清 miRNA表达情况，我们结合

了低密度基因芯片和 RT-qPCR两种技术。首先，用低密度基因芯片技术检测血清样本池，从而筛选出一批候选 miRNA；然后，再用 RT-qPCR 技术单独检测 160个肺腺癌患者和 160个健康对照的血清，从而验证候选miRNA。结果我们从总的 180

例肺腺癌患者和 180例健康对照的血清样本中发现 miR-103，miR-146a，miR-151-

3p，miR-221，miR-222和 miR-223这 6个miRNA在肺腺癌患者血清中明显上调，此结果暗示这些差异表达的 miRNA有可能成为肺腺癌患者血清 miRNA表达特征。进一步的分析结果显示，miR-146a，miR-222和miR-223这三个miRNA在 I/II期肺腺癌患者血清中的表达水平明显高于健康对照和其他肺部疾病对照，表明这三个miRNA可以作为新型的非侵入性肿瘤标志物，用以对高危人群和早期肺腺癌患者进行高灵敏度及高特异度地筛查。

另外，在此次研究中发现的肺腺癌患者血清中异常表达的 miRNA中，大多数都是与癌症相关的 miRNA。例如，miR-222曾被发现是在 TRAIL抵抗的非小细胞肺癌中上调最多的 miRNA之一[28]，并且也被证实其在非小细胞肺癌患者血清中表达明显增多[29]。miR-223也被发现在非小细胞肺癌患者血清中表达水平有所上升[30]，另外也有研究发现 miR-223在胃癌、结肠癌及胰腺癌等胃肠道肿瘤的肿瘤组织中表现为表达上调[31, 32]。与我们的结果一致，miR-146在非小细胞肺癌患者血清中也有表达上调的趋势[33]，不过其在肺癌中所起的功能还不甚明了，但是有研究发现其在 NIH/3T3细胞中可促进细胞的增殖及集落形成，据此推测 miR-146

可能在肺癌的发生发展中也起着一些重要的作用 [34]。为了进一步探明血清miRNA在肺腺癌中的作用及调节血清 miRNA表达的潜在机制，还需要更深层次的科学研究。还有一些miRNA，例如miR-155、miR-566以及miR-939等，曾被报道在肺腺

癌患者血清中表达上调，但是这几个 miRNA均为出现在我们的候选 miRNA中。Gao等[35]学者发现与健康对照相比，miR-155在肺腺癌患者血清中表达明显上升，但是其在肺腺癌患者的血清中表达水平比健康对照只升高了 2.034 倍。而在我们筛选候选 miRNA的第一阶段，只有当血清 miRNA表达水平在肺腺癌患者和健康对照之间相差至少 5 倍才算做差异表达 miRNA，因此我们筛选到的 11 个候选miRNA中不包括miR-155。Rani等人[36]发现miR-566和miR-939在肺腺癌患者血清中表达水平明显高于健康对照，但是比较肿瘤 TNM分期的四期，发现这两个miRNA只在 I期和 II期肺腺癌患者血清中表达上调，而在 III期肺腺癌患者血清中表达水平反而大幅降低。由于我们第一阶段用 Exqion高通量 RCP 技术筛选候选miRNA时，是用的 20个肺腺癌患者或 20个健康对照的血清混合而成的样本池，所以我们推测，可能是 III期肺腺癌患者的血清对样本池起到了稀释作用，导致样本池中miR-566和miR-939的浓度降低，从而未筛选到这两个miRNA。

近些年的研究报告显示，在各种体液中都曾检测到细胞外的 miRNA或循环miRNA，并且这些miRNA具有很多理想肿瘤标志物所需的具备的性质，比如可在富含核酸酶的体液中稳定存在，具有独一无二的序列，组织特异性表达等。越来越多的研究数据显示，有三种膜结构小囊泡中包含细胞外 miRNA，主要为凋亡小体、脱落的囊泡以及外泌体[37-39]。除了这些囊泡，细胞外 miRNA还可以独立存在于囊泡之外，单独与 AGO 蛋白相连，或包裹在高密度脂蛋白（High-Density

Lipoprotein，HDL）颗粒中[40-42]。目前主要有两个与循环 miRNA的输出和功能有关的理论：（i）它们主要由常规分泌及细胞死亡产生的微囊泡输出；（ii）它们是由细胞特异性分泌的，参与细胞间信号交流[43]。这两种理论均有可能是真实的，但还需要有更深入的研究去证实体内循环 miRNA起信号分子作用的理论。综上所述，miR-146a，miR-222和miR-223所组成的 miRNA 组具有足够高的

灵敏度和特异度从健康对照或其他肺部疾病中鉴别出肺腺癌。因此，该 miRNA 组 可以作为肺腺癌早期检测的一种新型的非侵入性肿瘤标志物，以及肺腺癌常规筛查的一种补充检测方法。5. 参考文献[1] Jemal A, Siegel R, Xu J, et al. Cancer statistics, 2010. CA: a cancer journal for

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类别：临床实验室生物安全管理及分子诊断技术与临床应用914880Analysis of Accuracy of Z scores in Non-invasive Prenatal Test for Fetal Trisomies 13, 18, and 21 That Employs the Ion Proton Semiconductor Sequencing PlatformYuan Tian1, Linlin Zhang1, Weifang Tian1, Jinshuang Gao1, Liting Jia2

1 Department of prenatal diagnosis laboratory of the Third Affiliated Hospital of

Zhengzhou University,Zhengzhou,China

2 Department of clinical laboratory of the Third Affiliated Hospital of Zhengzhou

University,Zhengzhou,China

Abstract

BACKGROUND: Non-invasive prenatal test is frequently used to screen trisomies

13,18 and 21 for prenatal diagnosis. However, compared with invasive testing, NIPT

should not be used to diagnose trisomies. The result of NIPT for a woman in most of the

genome-wide methods is calculated as a Z-score. The aim of this study was to find the

relation of Z-scores of NIPT and the accuracy of NIPT.

RESULTS: We evaluated 108 pregnant women with NIPT positive results that were

validated through karyotype analysis of amniotic fluid puncture by means of sequencing,

bioinformatics analysis, and follow-up. By using the Ion Proton Sequencing Platform, we

reported a performance evaluation of NIPT positive results at

the Third Affiliated Hospital of Zhengzhou University of Henan through

classifing Z sores into 3≤Z ＜ 5, 5 ≤Z<9 and Z≥9.Our study indicated that NIPT positive results with Z≥9 have a higher accuracy compared with positive results with 5≤ Z<9 and 3≤Z

＜5.

CONCLUSIONS: we find that Z-scores of NIPT results are related to accuracy of

NIPT.We reckons that the false positive results with 3≤Z＜5 may be resulted from the occurrence of CPM.

Key words: Non-invasive prenatal testing (NIPT); False positive; Confined placental

mosaicism(CPM); Trisomies

Introduction

Recently non-invasive prenatal test (NIPT) is frequently used to screen trisomies 13, 18

and 21 for prenatal diagnosis. Because of uncomplicated operation and avoiding

amniocentesis, NIPT is regarded as the first choice when pregnant women are

considerated about trisomies. NIPT is an attractive option for women because it is

procedurally safe for both mother and fetus, it can be conducted as early as ten weeks

gestation, and is highly accurate [1].However, NIPT should not be used to diagnose

compared with invasive testing[2]. Confined placental mosaicism(CPM) is one of the

impacts on confirmation of NIPT results, which can occur through a mitotic

nondisjunction event or through aneuploidy rescue[3].Confined placental

mosaicism(CPM) is known as a phenomenon that the cytogenetic abnormality, most

often trisomy, is confined to the placenta[4].Because of the exist of CPM, NIPT result

may be the false positive.

Recent NIPT technologies are predominantly based on next‐generation

sequencing(NGS)[5]. This enables rapid and effective clinical testing such as the IONA®

test which uses the Ion Proton semiconductor sequencing platform and has a turnaround

time, from the start of sample processing to a result, of 3 days[6].The result of a NIPT for

an individual woman in most of the genome-wide methods is calculated as a Z-score,

where the individual sample is compared with a control group of normal (diploid)

samples. In some researchs, a posterioririsk(PPR) calculator is used to more accurately

express the true likelihood of carrying a foetus with trisomy, and research shows that ,the

PPR is effectively independent under all conditions for Z-scores above 6 and a high PPR

for low a priori risks can only be reached at Z-scores>5[7].

According to the present study, we decided to report a performance evaluation of NIPT

positive results in our hospital through classifing Z sores into 3≤Z＜ 5, 5 ≤Z<9 and

Z≥9.Our study indicated that NIPT positive results with Z≥9 have a higher accuracy

compared with NIPT positive results with 3≤Z＜5 and 5 ≤Z<9.

Patients and methods

Patients Written informed consent was obtained from each patient. The study was a

retrospective analysis of NIPT positive results in singleton pregnancy from January 2015

to December 2017 at the Third Affiliated Hospital of Zhengzhou University of Henan

province, China. The patients tested for positive results through NIPT would be

validated through postpartum follow-up whose content contained the results of karyotype

analysis of amniotic fluid puncture, including trisomy13,18,and 21.In total, 108 pregnant

women with NIPT positive results were validated through karyotype analysis of amniotic

fluid puncture.

Sequencing Maternal peripheral blood (5 ml) was collected in an

ethylenediaminetetraacetic acid (EDTA) tube at the gestational age of 12+0 to 25+6

weeks. The blood sample was stored at 4°C immediately after collection. Plasma was

isolated within 8 hours with a two-step centrifugation protocol. Subsequently, samples

were delivered at −80°C. The cell-free DNA extraction, library construction, sequencing,

and bioinformatics analysis were performed according to the previous study[6] .

Bioinformatics analysis Sequencing data were analyzed by using the Ion Proton

semiconductor sequencing platform and software from Capitalbio Corporation. The

binary hypothesis Z-score of particular chromosomes in each sample was determined, as

reported previously[7].At last, to assess the fetal risk of T21, T18 and T13, the sample

with a Z-score ≥3 for these chromosomes was classified as positive. We classified the

positive samples into three groups according to the Z-score=5 and Z=9 among them.

Group 1 contained the samples whose NIPT results were 3≤Z＜5 and Group 2 contained

the samples whose NIPT results were 5≤Z＜9. Group 3 contained the samples whose

NIPT results were Z≥9 .

Karyotype analysis and amniotic fluid puncture Women with positive NIPT results

were recommended to receive karyotype analysis and amniotic fluid puncture for further

validation. The amniotic fluid puncture was performed as routinely described. The

karyotype analysis was performed according to the International System for Human

Cytogenetic Nomenclature guidelines[8].

Follow-up Follow-up was performed to NIPT positive cases via telecommunication. The

follow-up duration was 56 days after delivery.

Results

1. Patient characteristics

A total of 108 NIPT positive pregnant women who were validated through karyotype analysis and

amniotic fluid puncture, with a mean maternal age of 33.5 (19-48 years), were included in this

study. The gestational age was from 12 weeks to 25+6 weeks at blood sample collection. The

patient characteristics were listed in Table I.

Table I. Basic Characteristics of 108 NIPT Positive Pregnant Women.

Characteristics Numbers.Maternal age(years)≤20 120-24 525-29 3130-34 2135-39 3540-45 13≥45 2Mean 33.5Range 19-48Gestation at NIPT tests(weeks)12-12+6 413-13+6 914-14+6 515-15+6 816-16+6 1717-17+6 1418-18+6 1419-19+6 1720-20+6 521-21+6 622-22+6 223-23+6 524-24+6 025-25+6 2

2. NIPT positive results of Group 1 related with karyotype analysis and amniotic fluid

puncture

18 NIPT positive results in total were in Group 1(3≤Z＜ 5). The cases with false

positive results which were validated through karyotype analysis and amniotic fluid

puncture were 15, whose sensitivity was 83.33%. However, the true positive results

which were validated through karyotype analysis and amniotic fluid puncture were 3,

whose sensitivity was only 16.67%.The NIPT positive results in Group 1 were listed in

Table II. Through analyzing the data of the 3 true positive NIPT results, we found that

the fetal DNA fraction estimation for sample YC151439 and YC173711 was less than

4%. Besides, the fetal DNA fraction estimation for sample YC174141 was 6.4%,

which was less than 8%.

Table II. The 18 NIPT Positive Results in Group 1(3≤Z＜5)

No. Sample ID Z-scores NIPT results

Fetal karyotype

1 YC150550 3.330 T21 46,XN2 YC150816 4.342 T13 46,XN3 YC151439 3.643 T21 47,XN+214 YC161217 4.056 T13 46,XN5 YC163341 4.778 T13 46,XN6 YC164267 3.968 T13 46,XN7 YC165157 3.392 T21 46,XN8 YC165602 3.362 T18 46,XN9 YC167031 3.075 T21 46,XN10 YC171503 3.207 T21 46,XN11 YC173711 4.951 T21 47,XN+2112 YC174141 3.242 T21 47,XN+2113 YC175988 3.713 T21 46,XN14 YC175996 4.111 T18 46,XN15 YC176477 3.351 T18 46,XN16 YC176775 3.620 T21 46,XN17 YC177355 3.263 T21 46,XN18 YC177383 3.697 T13 46,XN

3. NIPT positive results of Group 2 related with karyotype analysis and amniotic fluid

puncture

A total of 34 NIPT positive results were in Group 2(5≤Z＜9). The cases with false positive results

which were validated through karyotype analysis and amniotic fluid puncture were 4, whose

sensitivity was 11.76%. However, the true positive results which were validated through

karyotype analysis and amniotic fluid puncture were 30, whose sensitivity was 88.24%.The NIPT

positive results in Group 2 were listed in Table III.

Table III. The 34 NIPT Positive Results in Group 2(5≤Z＜9)

No.

Sample ID Z-scores NIPT results

Fetal karyotype

1 YC150539 5.570 T21 47,XN+212 YC150431 5.960 T18 47,XN+183 YC150725 7.463 T21 47,XN+214 YC152284 8.249 T21 47,XN+215 YC153184 8.532 T21 47,XN+216 YC160356 8.830 T21 46,XN7 YC161880 8.188 T21 47,XN+218 YC163276 5.467 T18 47,XN+189 YC163808 6.104 T21 47,XN+2110 YC164065 5.219 T18 47,XN+1811 YC164811 8.489 T21 47,XN+2112 YC165074 6.677 T21 47,XN+2113 YC165729 5.944 T21 47,XN+2114 YC166180 6.150 T21 47,XN+2115 YC166202 6.220 T18 47,XN+18

16 YC166253 5.931 T2146XX+21 ， der(14;15)(q10,q10)

17 YC166422 8.945 T13 46,XN18 YC167160 5.710 T18 46,XX/47,XX+18(28:2)19 YC167331 7.790 T21 47,XN+2120 YC168025 6.840 T21 47,XN+2121 YC170096 8.280 T21 47,XN+2122 YC171234 6.119 T21 47,XN+2123 YC171535 7.273 T21 47,XN+2124 YC171540 6.019 T21 47,XN+2125 YC173331 5.330 T21 47,XN+2126 YC173539 7.514 T21 47,XN+2127 YC173759 6.834 T21 47,XN+2128 YC175051 6.908 T21 47,XN+2129 YC175492 8.646 T18 47,XN+1830 YC175694 6.704 T13 47,XN+1331 YC176740 7.803 T21 47,XN+2132 YC177345 5.178 T21 46,XN33 YC176881 5.566 T21 47,XN+2134 YC177644 8.523 T21 47,XN+21

Through analyzing the data of the 4 false positive results, we found that sample

YC167160 was validated as CPM through karyotype analysis and amniotic fluid puncture

(Table III). Sample YC160356 was possibly affected by the chromosome duplication

from maternal fragment (Fig 1). The false NIPT positive results of sample YC166422 and

YC177345 might be resulted from a small period of chromosome duplication(Fig 2 and

Fig 3).

Fig 1. The Distribution Curve of Z-score from Chromosome 21 of Sample YC160356

Fig 2. The Distribution Curve of Z-score from Chromosome 13 of Sample YC166422

Fig 3. The Distribution Curve of Z-score from Chromosome 21 of Sample YC177345

4. NIPT positive results of Group 3 related with karyotype analysis and amniotic fluid

puncture

Totally 56 NIPT positive results were in Group 3(Z≥9).The cases with false positive

results which were validated through karyotype analysis and amniotic fluid puncture

were 1, whose sensitivity was 1.88%. For the sample YC175377, the unique sample

which owned false positive results, we found that the pregnant woman suffered from

diapause of one fetus in bigeminal pregnancy within 4 weeks via follow-up. Besides, the

true positive results which were validated through karyotype analysis and amniotic fluid

puncture were 55, whose sensitivity was 98.12%. The NIPT positive results in Group 3

were listed in Table IV.

Table IV. The 56 NIPT Positive Results in Group 2(Z≥9)

No.

Sample ID Z-scores NIPT results

Fetal karyotype

1 YC151198 11.203 T21 47,XN+212 YC151297 11.605 T21 47,XN+213 YC151374 9.306 T21 47,XN+214 YC151424 19.541 T21 47,XN+215 YC151577 14.882 T21 47,XN+216 YC151725 12.367 T21 47,XN+217 YC152585 18.082 T21 47,XN+218 YC153464 10.126 T21 47,XN+219 YC153533 11.864 T21 47,XN+2110 YC160344 14.298 T21 47,XN+2111 YC161058 10.8 T21 47,XN+2112 YC161066 21.457 T21 47,XN+2113 YC161368 13.275 T21 47,XN+2114 YC162688 9.707 T21 47,XN+2115 YC162822 16.21 T18 47,XN+1816 YC162943 11.347 T21 47,XN+2117 YC163223 13.694 T21 47,XN+2118 YC163261 13.627 T21 47,XN+2119 YC163310 13.977 T18 47,XN+1820 YC163400 11.603 T13 47,XN+1321 YC164204 10.546 T21 47,XN+2122 YC164355 11.135 T21 47,XN+2123 YC164722 13.955 T21 47,XN+2124 YC164999 13.661 T21 47,XN+2125 YC165003 11.899 T18 47,XN+1826 YC165723 10.192 T21 47,XN+2127 YC165823 10.346 T18 47,XN+1828 YC165859 11.962 T18 47,XN+1829 YC166626 9.374 T13 47,XN+1330 YC167544 11.253 T18 47,XN+1831 YC168149 11.004 T21 47,XN+2132 YC170133 12.085 T21 47,XN+2133 YC170411 12.536 T21 47,XN+2134 YC170724 10.017 T21 47,XN+2135 YC170829 12.456 T21 47,XN+2136 YC170974 11.555 T21 47,XN+2137 YC171134 9.885 T21 47,XN+2138 YC171423 11.41 T21 47,XN+2139 YC172015 18.693 T21 47,XN+2140 YC172946 20.224 T21 47,XN+2141 YC173326 9.674 T21 47,XN+2142 YC173370 17.431 T21 47,XN+2143 YC173573 26.587 T21 47,XN+21

44 YC173742 9.054 T21 47,XN+2145 YC174011 13.767 T21 47,XN+2146 YC174110 11.488 T21 47,XN+2147 YC174589 16.765 T21 47,XN+2148 YC175377 12.367 T18 46,XN49 YC175406 11.424 T21 47,XN+2150 YC175583 9.772 T21 47,XN+2151 YC175824 13.609 T21 47,XN+2152 YC175903 12.095 T21 47,XN+2153 YC176469 11.443 T18 47,XN+1854 YC177386 25.448 T18 47,XN+1855 YC176915 12.225 T21 47,XN+2156 YC178093 18.509 T18 47,XN+18

Discussion

NIPT has been widely used for the prenatal screening of T21, T18 and T13 in the last few

years. Up to now although PPR calculator reckoned that a high PPR for low a priori risks

can only be reached at Z-scores>5[7], it is still lacking large scale clinical studies to

validate the hypothesis. The present study including 108 NIPT positive cases was

performed to investigate the relation of NIPT Z-scores and accuracy of the diagnosis of

trisomy13, 18, and 21 respectively.

Through the present study, we find that, it seems that the sample with Z≥9 owns a higher

accuracy than the sample with 3≤Z＜5 and 5≤Z＜9. Although the true positive rate in

Group 2(88.24%) is apparently higher than the true positive rate in Group 1(16.67%), it

cannot be used to diagnose because of its relative high false positive rate (11.76%).

However, the positive results with Z≥9 have a positive significance to diagnosing

trisomies 13, 18 and 21. The true positive rate with Z≥9 is approximately 100% except

for the particular case like sample YC175377. Maybe the difference of them is resulted

from the occurrence of confined placental mosaicism(CPM).There are a lot of biological

reasons for discordant results that can be of either fetal or maternal origin. Contributing

fetal factors include insufficient or absent fetal fraction, confined placental mosaicism

and the presence of a vanishing twin[9].For example, Hall AL et al ever reported a case

that confirmed positive cell-free fetal DNA testing for trisomy 13 reveals confined

placental mosaicism[10].Chen C et al found a pregnancy with discordant fetal and

placental chromosome 18 aneuploidies revealed by invasive and noninvasive prenatal

diagnosis[11].According to above studies, we reckons that the occurrence of CPM may

slightly promote Z-score of NIPT, only making it beyond 3 but below 5.

In addition, the fetal DNA fraction estimation is of importance for accuracy of the NIPT

results. The circulating cell-free DNA (cfDNA) in a pregnant woman is a mixture of

predominant maternal DNA derived from the hematopoietic system of the mother and

fetal DNA released through the apoptosis of cytotrophoblast cells during fetal

development[12]. The fetal DNA concentration less than 4% in a maternal plasma sample

would suggest a potential issue present in the quality control (QC) step[13]. Because the

limited amount of fetal DNA molecules to be detected and analyzed may give rise to a

false negative result, we deduce that Z-scores of sample YC151439, YC173711 and

YC174141 are less than 5 because of their low fetal DNA fraction estimation.

On the other hand, maternally derived chromosomal abnormalities (probably also the

submicroscopic ones) are a rare case of maternal occult malignancyand usually cause

false-positive results[14].Through our study, the hypothesis is validated by the false

positive NIPT result from sample YC160356(Fig 1).

Conclusions

NIPT technique is feasible for the prenatal screening of T13,T18 and T21 with higher

sensitivity and specificity compared with conventional methods. However, Z-scores of

NIPT results are closely related to accuracy of non-invasive prenantal test that emloys

the Ion Proton semiconductor sequencing platform. NIPT results with low Z-scores

should not be used to diagnose compared with invasive testing. In addition, the relation

of Z-scores of NIPT and CPM is not approved by large scale clinical studies.

Acknowledgment

Not applicable.

Funding

The study was supported by Capitalbio Corporation and the Third Affiliated Hospital of

Zhengzhou University.

Availability of data and materials

The datasets obtained in this study are available from the corresponding author.

Abbreviations

NIPT Non-invasive prenatal test

CPM Confined placental mosaicism

EDTA Ethylenediaminetetraacetic acid

cfDNA The circulating cell-free DNA

QC quality control

Authors’contributions

Yuan Tian performed the bioinformatics analysis and drafted the manuscript. Linlin

Zhang performed Karyotype analysis and amniotic fluid puncture and follow-up.

Weifang Tian performed recruited the patients, provided blood samples, clinical data for

each partcipant and helped to draft the manuscript. Jinshuang Gao performed sequence

of NIPT. Liting Jia performed statistical analysis and helped to draft the manuscript.

Ethics approval and consent to participate

The study was approved by the Ethics committee of the Third Affiliated Hospital of

Zhengzhou University, where it was conducted. Each patient signed an informed consent

prior to the study enrollment.

Consent for publication

All the participants accepted with the initial written informed consent the possibility that

the results of this study could be published.

Competing interests

The authors declare that they have no competing interests.

Contibutor Information

Yuan Tian, Email:741603184@qq.com

Linlin Zhang, Email: linlinzlynn@163.com

Weifang Tian, Email: weifang0524@126.com

Jinshuang Gao, Email: gaojs10@163.com

Liting Jia, Email: jialt@163.com

References:

[1]. Minear, M.A., et al., Global perspectives on clinical adoption of NIPT. Prenat

Diagn, 2015. 35(10): p. 959-67.

[2]. Lau, T.K., et al., Non-invasive prenatal testing for fetal chromosomal

abnormalities by low-coverage whole-genome sequencing of maternal plasma DNA:

review of 1982 consecutive cases in a single center. Ultrasound Obstet Gynecol, 2014.

43(3): p. 254-64.

[3]. Mardy, A. and R.J. Wapner, Confined placental mosaicism and its impact on

confirmation of NIPT results. Am J Med Genet C Semin Med Genet, 2016. 172(2): p.

118-22.

[4]. Kalousek, D.K. and M. Vekemans, Confined placental mosaicism. J Med Genet,

1996. 33(7): p. 529-33.

[5]. Vrachnis, N., N. Vlachadis and G. Creatsas, DNA sequencing versus standard

prenatal aneuploidy screening. N Engl J Med, 2014. 371(6): p. 578.

[6]. Papageorghiou, A.T., et al., Clinical evaluation of the IONA test: a non-invasive

prenatal screening test for trisomies 21, 18 and 13. Ultrasound Obstet Gynecol, 2016.

47(2): p. 188-93.

[7]. Sikkema-Raddatz, B., et al., NIPTRIC: an online tool for clinical interpretation of

non-invasive prenatal testing (NIPT) results. Sci Rep, 2016. 6: p. 38359.

[8]. Gonzalez, G.J. and J.P. Meza-Espinoza, Use of the International System for

Human Cytogenetic Nomenclature (ISCN). Blood, 2006. 108(12): p. 3952-3; author reply

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415-423.

[10]. Hall, A.L., et al., Positive cell-free fetal DNA testing for trisomy 13 reveals

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generation sequencing tests for common fetal aneuploidies. Prenat Diagn, 2013. 33(7): p.

667-74.

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类别：临床实验室生物安全管理及分子诊断技术与临床应用906946 获得性易栓症患者的基因突变排除诊断李正民 1,2 祁新坤 1,2贺婧娴 1,2 陈瑞飞 1,2李涛 1,2 宫璀璀 3 吕金利 4 郭思 1,2 许泼实

1,2

(1. 阜外华中心血管病医院检验科，河南郑州，450000；2.河南省人民医院检验科，河南郑州，450000；

3.中国人民解放军第 153中心医院检验科，河南郑州，450000；4.中国人民解放军第 153中心医院普外科，河南郑州，450000）

摘要 目的 对多发性下肢静脉栓塞患者及其家系进行相关检查和基因诊断，分析其所患易栓症类型以及与遗传的相关性。方法 对该家系 4人用发色底物法对抗凝血酶活性（AT:A）、蛋白 C活性（PC:A）和蛋白 S活性（PS:A）分别进行检测，Western blot 检测血浆中的抗凝血酶含量和分子量，抽提患者外周血全基因组DNA，PCR 扩增抗凝血酶的 7个外显子及其侧翼序列，用直接测序法对患者的扩增产物进行测序分析和基因突变分析。结果 该患者的 AT:A为 52.1%、AT含量正常，AT基因序列的突变分析未发现突变位点，其他检测结果正常；该家系其他成员的 PC:A 、PS:A 、AT:A及 AT含量均正常。结论 根据相关检测结果及基因序列分析，结合患者以往有下肢骨折外伤史和手术史及长时间熬夜上网习惯，该患者诊断为获得性易栓症。关键词 获得性易栓症，遗传性易栓症，抗凝血酶缺陷症，抗凝血酶

易栓症是指易发生静脉血栓栓塞症倾向的一类疾病，分为遗传性易栓症和获得性易栓症。遗传性易栓症常见有抗凝血酶（antithrombin, AT）缺陷、蛋白 S缺陷和蛋白 C缺陷、FV Leiden突变和凝血酶原 20210A突变等；获得性易栓症常见诱因有年龄增加、长时间止动、创伤及手术史、妊娠期或产褥期以及长期口服避孕药或激素等，有些疾病（如：抗磷脂综合症、获得性抗凝蛋白缺乏、获得性凝血因子升高、糖尿病、肾病综合症等）也易诱发易栓症。易栓症的发生可以是单因素导致发

病，亦常常是几种诱因共同作用导致发病，可以是多种获得性诱因或遗传性因素的作用，也可以是获得性诱因和遗传性因素的共同作用。1.资料与方法1.1患者及家系资料

患者，男，汉族，20岁，几年前有右侧下肢骨折及手术史，下肢康复后多次出现下肢静脉血栓，近来于 2011年底因右侧下肢胀痛入院治疗，血管超声诊断为右下肢深静脉血栓，2012年 8月再次因右下肢深静脉血栓入院行溶栓治疗，出院后给予华法林维持治疗半年，定期监测凝血机制。患者家系其他成员无静脉血栓史，家族史阴性，患者有长期熬夜上网玩游戏的习惯。家系图见图 1。

图 1 易栓症患者家系图1.2相关检测指标：在获得该家系所有成员的知情同意后，对家系成员 4人分别采用负压静脉采血（枸橼酸钠抗凝管采血 2.7ml2管、干燥管采血 5ml、EDTA管采血2ml。）和留取晨尿标本各 10ml。血样立即 4℃ 3000r/min离心 10分钟（除 EDTA

管），样本分别进行尿液分析（长春迪瑞试剂，长春迪瑞 H800 型尿液分析仪）、凝血四项(含 PT、APTT、TT、FIB；)和 D-D二聚体检测（法国 Stago 公司试剂，法国 Stago 公 司 STA-R Evolution 型全自动血凝仪）、血液细胞分析（日本Sysmex2100 型血球仪）、蛋白 C活性（PC:A）和蛋白 S活性(PS:A)及 AT活性检测（采用发色底物法，法国 Stago 公司试剂盒，法国 Stago 公司 STA-R Evolution 型全自动血凝仪）、肝功能、肾功能、血糖及同型半胱氨酸(HCY)含量检测(四川迈克生物科技股份有限公司试剂，日本日立 7600 型全自动生化分析仪)、狼疮抗凝物（LA）及抗心磷脂抗体检测（北京贝尔试剂盒，德国 Biotek酶标仪）等。

1.3 Western blot检测血浆中 AT含量：从-80℃冰箱取出冻存的该家系各成员枸橼酸钠抗凝血浆、正常人对照血浆共 5份，将血浆进行 20 倍稀释（用磷酸盐缓冲液稀释），再与上样缓冲液 3：1混合后放入 100℃沸水中煮沸 5min。加样后电泳（10%SDS-PAGE，60V,55mA）3.5h，经湿转法(60V,160mA)电泳 3h将蛋白条带转移至硝酸纤维素膜上，再用 5%脱脂奶粉（脱脂奶粉 0.5g，TBST 10ml）封闭 1h, 用一抗（Antithrombin Ⅲ Rabbit Monoclonal Antibody，美国 EPIT MICS 试剂，An

Abcam 公司）孵育 2h，经彻底清洗后用二抗（HRP标记的 goat anti-rabbit IgG，上海生工公司分装）孵育 2h。彻底清洗后用 DAB显色液（北京中杉金桥）显色 10

分钟。同时进行-actin的内参照显色，阳性条带呈现棕色后用水终应。应用美国 ALPHA 凝胶成像系统进行扫描拍照。蛋白质分子量标记采用彩色预染蛋白质Marker(11-180kD)。1.4 全基因组 DNA 抽提：取该家系成员外周血（枸橼酸钠抗凝）各 200ul，使用欧盟 Fermentas DNA提取试剂盒提取全基因组DNA，严格按照试剂说明书操作提取。1.5 PCR 扩增及产物纯化：第 1、2、4、5、6、7外显子的扩增引物序列参照文献报道 [1]，第 3外显子的引物序列使用 Primer5引物设计软件设计，引物信息见表1。PCR 总体系为 25ul：引物终浓度为 0.2umol/L，模板 DNA 50ng，Taq 酶为1.25U（北京 TIANGEN）， dNTP 终浓度为 0.25mmol/L，MgCl2 终浓度为1.5mmol/L。反应条件：95℃预变性 5 min，然后 95℃ 30s，57℃退火 30s(第 3外显子采用 62℃退火，第 5外显子采用 60℃退火)，72℃延伸 30s,30个循环，72℃

延伸 10min。取 PCR产物 80ul，加入 20ul loading buffer(4:1)混合,进行琼脂糖电泳（18g/L琼脂糖，含溴化乙锭）30min，应用美国 ALPHA 凝胶成像系统进行扫描拍照，图 2。将患者的相应电泳条带在 254nm紫外灯下切取，用 QIAquick Extracion

Kit试剂盒进行 PCR产物回收、纯化（美国QIAGEN 公司）。表 1 AT基因 PCR引物序列及扩增区域

外显子 引物序列（5’→3’） 扩增片段 位置

Exon1 F: CAGCCCTGTGGAAGATTAGC 152bp 581-732R: CAAAGGTGTTGGAGGTCATTC

Exon2 F: TAACTTGGCATTTTGTCTCC 418bp 2918-3335R: CTGGGCAGAAGACCTTG

Exon3 F: CACCAAACCCACCACCATTT 327bp 5806-6132R: GCTGTTTCTCCACCTCCTCA

Exon4 F: TTGAATAGCACAGGTGAGTAGG 234bp 6920-7153R: GCTGAAGAGCAAGAGGAAGT

Exon5 F: TGTGATTCTCTTCCAGGGC 442bp 7896-8337R: GGTGGAGAAGGGAGGAAAC

Exon6 F: CAACTTTCTCCCATCTCACAA 148bp 10290-10431R: TTTCTGTACCCTAAGAGAGTGG

Exon7 F: GTTTTATTCCCATGTGACCTG 211bp 13750-13960R: AAGAACATTTTACTTAACACAAGG

图 2 患者AT基因 7个外显子的扩增产物电泳图1.6 AT基因测序及突变分析：PCR纯化后产物委托上海生工生物工程有限公司进行正向和反向测序（测序仪器为美国 ABI 公司 3730xl基因分析仪，试剂为 BigDye

terminator v3.1，测序图谱分析采用 Chromas软件。）。将患者 AT基因测序结果用BLAST在线工具与 AT基因标准序列(Genbank:X68793.1)进行比对，进行基因突变分析。1.7 排除 FV Leiden突变和凝血酶原 G20210A突变：对患者进行 FV Leiden突变检测 ： 引 物 为 F, 5’CCATTATTTAGCCAGGAGACC3’ ， R,

5’ATTGGTTCCAGCGAAAGC3’；对外显子 10及侧翼序列进行扩增（退火温度60℃）、测序分析。同时进行凝血酶原 G20210A 突变检测：引物为F,5’TCTAGAAACAGTTGCCTGGC3’, R,TCCAGTAGTATTACTGGCTC3’；对外显子 14及侧翼序列进行扩增（退火温度 53℃）、测序分析。2.结果

2.1相关检测结果：患者检测结果：AT:A 52%, PS:A 121%,PC:A 104%,PT 12.4s,INR

1.02,APTT 32.1s,TT 14.6s,FIB 2.43g/L；血液细胞分析、肝功能、肾功能等无明显异常，血糖、尿液分析、D-D二聚体、抗心磷脂抗体、LA、同型半胱氨酸等检测结果均正常。该家系 4人中仅有患者表现为 AT:A的降低，其他成员的 AT、蛋白 C

和蛋白 S活性等检测结果均正常。Western blot检测结果显示：该家系 4名成员的 AT蛋白在 58kD区域处存在与

正常对照分子量一致的特异性条带，AT蛋白条带阳性强度经美国 ALPHA 凝胶成像系统进行灰度值扫描与正常人条带扫描结果接近（见图 3）。说明该患者（Ⅱ 2）的血浆中AT含量和分子量正常，仅表现为AT活性降低。

图 3 该家系AT蛋白Western blot检测结果2.2 测序结果及突变分析：将患者 AT基因 7个外显子的测序结果与 AT基因标准序列(Genbank:X68793.1)进行比对分析，用 BLAST工具将测序序列转化成氨基酸序列，与美国国家生物信息中心（NCBI）公布的 AT氨基酸序列进行比对，未发现AT基因突变位点和氨基酸异常序列。2.3 FV Leiden突变和凝血酶原 G20210A突变检测结果：对先症者的 FV基因第 10

外显子和凝血酶原基因第 14外显子进行 PCR 扩增、产物测序，未发现 FV Leiden

突变和凝血酶原G20210A突变。3. 讨论

AT是一种由肝细胞合成分泌的糖蛋白，分子量为 58KD小分子蛋白，编码 AT

蛋白的基因全长 14206 bp（ GenBank: X68793.1），位于染色体 1q23-25，共有 7个外显子、6个内含子。AT的蛋白结构较为复杂，由 464个氨基酸组成，有 3个 β 折叠（包括 A、B、C）、9个 a螺旋和一个反应中心环（RCL），包含了 3个二硫键和 4种糖基化位点。AT能够灭活凝血酶、FXa等因子，具有抗凝血作用，与肝素

结合后可使抗凝血作用增加数千倍以上[2]。有研究报道[3-5]:AT有两个重要的功能区，一个是位于羧基（C）端的反应位点 ,AT 的反应位点在 P1（Arg393）位和P1’(Ser394)位，P1 通过与凝血酶活性中心的 Ser结合，形成稳定的AT-凝血酶复合物，而灭活凝血酶; 另一个是位于氨基（N）端的肝素结合区。据研究发现AT在血液中的浓度较低，此时 AT的抗凝活性较小，但在有肝素分子存在的情况下，能够与肝素中的戊糖（pentasaccharide）序列结合，AT的构象发生改变，反应中心环（RCL）暴露，能够最有效的发挥抗凝作用[6-11]。大多数情况下，易栓症患者的发病常常是受到了获得性因素的影响，如弥漫性血管内凝血（DIC）、肝脏疾病、肾病综合症、抗磷脂综合症、妊娠、手术、创伤、长期慢性腹泻、口服避孕药及雌激素、恶性肿瘤等[12-18]。获得性易栓症亦指获得性血栓危险因素或获得性易栓状态。各种不同类型手术静脉血栓的发生率差异较大，尤其是骨外科和神经外科手术，如膝关节和髋关节手术、下肢和骨盆骨折、脑外伤等的血栓发生率为最高；瘫痪、久病在床、术后卧床、石膏长期固定的病人，由于血流淤滞，也易发生静脉血栓；长时间飞行后也易发生静脉血栓的现象称为“经济舱综合症”。此外，遗传性因素方面，也可引起家族型易栓症，如：AT基因突变可以导致 AT蛋白的分泌障碍、胞内滞留而不能发挥抗凝作用，还可以使 AT的反应位点异常变异而致活性降低，使AT的肝素结合位点异常变异而致与肝素亲和力降低[19-21]。易栓症的获得性和遗传性因素并存时，血栓形成的危险性明显增高。该患者AT基因测序结果显示：未发现 AT基因突变、FV Leiden突变和凝血酶

原 G20210A突变，PS:A、PC:A检测正常可排除 PS和 PC蛋白异常的影响，肝肾功能、血糖、凝血四项、抗心磷脂抗体、LA、同型半胱氨酸和 D-D二聚体等均正常，仅表现为 AT:A活性降低，而患者有创伤史、手术史、长期止动习惯是获得性易栓症发病的一些重要诱因。该患者家族史阴性，家系其他成员经相关检测均正常，可以排除遗传性易栓症的影响，该患者诊断为获得性易栓症，给予抗凝、溶栓治疗，康复后定期监测凝血机制，适当服用抗凝药物进行预防性用药。

综上所述，该文检测了一例 AT:A减低而 AT基因序列检测未发现 AT基因突变的患者，排除了遗传性因素的影响。本文从基因诊断的角度做了深入研究发现 :

获得性易栓症患者有时也可伴有 AT:A降低，而无 AT基因突变, AT:A降低不仅仅存在于遗传性易栓症。获得性和遗传性 AT缺陷症的诊断应从家系调查、相关检查和基因水平进行判断，该文为以后的易栓症相关研究提供了相关资料和依据。参考文献：[1] 傅启华，许先国，丁秋兰等. 抗凝血酶基因 13389G缺失导致的Ⅰ型抗凝血酶缺乏症[J].中华血液学杂志，2002，23（11）：588-590.

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类别：临床实验室生物安全管理及分子诊断技术与临床应用908747 不孕不育者SAT法和尿液快速检测法检测淋球菌比较

包杰*，杨永俊河南省人口和计划生育科学技术研究院 河南 郑州 450002

摘要：目的 比较实时荧光核酸恒温扩增技术（SAT）和尿液快速检测法在淋球菌（NG）检测中的诊断价值，以选择更为准确、快速、实用的临床检测方法。方法 采集本院就诊的不孕不育对象 200例,每例分别进行 SAT 法和尿液快速检测法检测，每例另取 1份分泌物拭子标本做分离培养金标准对照用。结果 SAT法阳性率为22.00 % ，快速法阳性率为 26.00 %，其中疑似标本 SAT法与快速法阳性率分别为39.00 %、44.00 %，无症状标本 SAT 法与快 速法阳性率分别为 5.00 %、8.00

%。SAT法的敏感性和特异性分别为 100.00 % 、97.50 %，尿液快速法的敏感性和特异性分别为 100.00 % 、92.50 %，SAT法与尿液快速法的敏感性均较好，SAT法的特异性高于尿液快速法。结论 NG在不孕不育人群中隐性感染不容忽视，加强就诊对象NG的筛查、诊治和预防，确保优生优育。SAT 技术精准、特异，具有较高的临床实用性，是适合实验室诊断的检测方法。关键词：不孕不育；淋球菌；实时荧光核酸恒温扩增技术（SAT）；尿液快速检测法Comparison of Simultaneous Amplification and Testing and Rapid urine test in the

Detection of Neisseria gonorrhoeae among Infertility crowd

BAO Jie*, YANG Yong-jun

*Henan Province Research Institute for Population and Family Planning, Zhengzhou,

Henan450002,China

Corresponding author: BAO Jie, E-mail: 905293917@qq.com

Abstract:Objective Compare real-time fluorescence nucleic acid thermostatic

expansion technology (SAT) and rapid test urine of the diagnostic value in neisseria

gonorrhoeae examination, to select the more accurate, rapid and practical method for

clinical testing. Methods 200 infertile patients were collected from our hospital. Each

case was examined by SAT and urine rapid test, and 1 samples of secretion swabs was

used for the separation and culture of gold standard as a control group. Results SAT

method of positive rate was 22.00 %, rapid test urine positive rate was 26.00 %, Among

them, the positive rate of suspected specimen SAT and rapid method were 39% and 44.00

% respectively, asymptomatic specimens SAT method and rapid method positive rate

were 5.00 %, 8.00 % respectively. The SAT method, the sensitivity and specificity of

100%, 97.50 % respectively, urine rapid method of sensitivity and specificity were

100.00 %, 92.50 %, the SAT method and urine rapid method has good sensitivity and the

specificity of the SAT method is higher than the urine rapid method. Conclusion

Neisseria gonorrhoeae recessive infection should not be ignored in infertility population,

strengthen medical patient NG screening, diagnosis and treatment, and prevention, to

ensure the stirpiculture. SAT technique is accurate, specific and has high clinical

practicability. It is a suitable method for laboratory diagnosis.

Key words:Infertility; Neisseria gonorrhoeae; Simultaneous amplification and testing;

Rapid urine test

作者简介：包杰，副主任技师，本科，从事临床检验通讯作者：包杰，E-mail:905293917@qq.com

淋病是目前发病率较高的性传播疾病(STD)之一，它主要通过性接触传播由淋病奈瑟菌(NG)引起的泌尿生殖道化脓性感染，对人体生殖系统的健康造成了极大危害，孕龄女性一旦感染，容易引起输卵管堵塞、不孕、宫颈炎、流产、新生儿脓漏眼病等。据调查，不孕不育人群中不典型淋病症状者呈增长趋势，早期诊断，特别是对无症状患者的检出是控制淋球菌传播及感染的关键，孕前及时进行检测，并给予检测结果呈阳性患者治疗，降低感染几率，是实现优生优育、保证新生儿健康的重要手段[1]。

不典型淋病患者主要依靠实验室检查帮助诊断，目前实验室对淋球菌检测有直接涂片、分离培养、免疫荧光、自检卡等多种方法，分析时间长短不一，检出率高低不等，由于试剂盒制备、实验室条件及操作技术等各方面的差异, 其敏感性和特异性差别较大，其存在的假阳性和假阴性是困惑各实验室的普遍现象，且常用淋球菌检测的都是患者的泌尿生殖道分泌物标本，容易增加患者的痛苦、降低检测的依从性，因此，寻找准确、快速、实用的检测方法有着重要的意义[2]。为了找到可靠的淋病诊断方法，本研究选择了本院前来就诊的不孕不育对象 200 例，以尿液作为样本同时应用快速检测法和 SAT 技术进行 NG 的检测，对这 2 种方法的特异性、敏感性等指标来综合分析各自优缺点，以期为实验室选择正确的检测方法，为临床提供可靠的诊断依据，现报道如下。

1 资料与方法1.1 样本来源 选择 2017年 1～6月来本院门诊就诊的不孕不育人群有尿道口红肿、脓性分泌物明显、尿急尿频尿痛等症状的疑似病例 100例，无症状者 100例，年龄20～35岁。 要求所有人员在标本采集的前 7～10 d ，禁止采用抗生素药物，所有人员在身体方面无差异。1.2 样本采集 每个检查对象取 1 份尿道（/阴道）分泌物拭子和 2 份尿液标本。1.2.1 拭子标本采集 用特制无菌棉拭子伸入女性宫颈口或男性尿道口 1～2 cm，轻轻旋转并停留 10 s后取出，立即保温送检。1.2.2 尿液标本采集 1份初段尿标本用于淋球菌快速检测；另一份初段尿 1 mL与 1

mL 尿液保存液（SAT 试剂盒提供）混合置于 EP 管中-20 ℃冷冻储存，用于 SAT

检测。

1.3 试剂与方法

1.3.1 试剂 尿液淋球菌快速检测试剂盒由郑州安图绿科生物有限公司提供； SAT

法恒温扩增检测试剂盒（NG-RNA ）由上海仁度生物科技有限公司提供，TL 988

PCR 仪由西安天隆科技有限公司提供； 另购进郑州人福博赛生物技术有限责任公司生产的巧克力培养基做分泌物分离培养对照用。1.3.2 方法

1.3.2.1 尿液淋球菌快速检测按照试剂盒的说明进行操作，要求 1 h内完成。取 2

mL 初段尿加入反应试管内，垂直滴加 3滴试剂，塞紧管塞轻轻摇匀，放置 5 min

后再轻轻摇匀 1 次，观察反应管内尿液上部形成气泡情况。

1.3.2.2 SAT预留标本检测按照试剂盒说明书操作，设置阴、阳性对照。取 100 μL

核酸提取液与 400μL 加有尿样保存液的预留样本混匀，60℃ 恒温 5 min ，室温放置 10 min ，之后将样本转移到半自动核酸提取仪上提取 RNA，完成后吸取 30μL

含磁珠的检测液至 8 连管中 ，预热后加酶液 10μL ，42 ℃实时恒温扩增 40 min

[3]。

1.3.2.3 分离培养法取分泌物立即接种于巧克力琼脂培养基 , 置 5％CO2 环境中 37

℃培养 24～48 h，根据菌落形态进行鉴定，取典型菌落涂片染色镜检为革兰阴性双球菌者为阳性。1.4 统计学分析 尿液淋球菌快速法和 SAT法检测结果采用 SPSS13.0统计学软件中的配对2检验进行统计学分析，结果以率表示，P＜0.05 表示差异具有统计学意义。2 结 果2.1 结果判断

2.1.1 尿液淋球菌快速法是利用淋球菌侵入尿道上皮细胞 ,产生细菌生化酶, 加入试剂与含有此酶的尿液产生生化反应产生气泡来判断结果， 若液面上部形成一层气泡或液面上层管壁形成一圈细小的气泡者判读为阳性， 20 min 后结果无效。2.1.2 SAT 法检测结果根据实时荧光信号的出现时间和强度，结合阴、阳性对照对检验结果进行判定。dt≤35的样本为阳性；dt为40或无数值的样本为阴性；35 <dt

<40 的样本建议重新检测，结果dt <40 的样本为阳性（dt 为阈值线与样本曲线交点的横坐标读数）。2.1.3 以淋球菌培养法为金标准，计算SAT检测尿液样本及拭子样本的灵敏度及特异性。灵敏度=真阳性例数/(真阳性＋假阴性)例数×100 ％，特异度=真阴性例数/(真阴性＋假阳性)例数×100 ％。2.2 尿液淋球菌快速法和SAT 检测结果比较2.2.1 200 例受检者中尿液淋球菌快速法阳性率为 26.00 %，NG-SAT法阳性率为22.00 %，其中 100例疑似标本尿液快速法与 SAT法阳性率分别为 44.00 % 、39.00

%，100例无症状标本尿液快速法与 SAT阳性率分别为 8.00 % 、5.00 %，尿液快速法阳性率均高于 SAT法（P＜0.05），见表 1。

表 1 两种方法检测淋球菌阳性率对照统计表[n(%)]

标本类型 （n） 尿液淋球菌快速法 NG-SAT 法2值 P值阳性 阴性 阳性率(%) 阳性 阴性 阳性率(%)

疑似标本 100 44 56 44.00 39 61 39.00 3.732 0.047无症状标本 100 8 92 8.00 5 95 5.00 4.130 0.038合计 200 52 148 26.00 44 156 22.00 3.021 0.049

2.2.2 200 例受检者中，SAT法与分离培养法检测NG时有6例结果不一致，尿液快速法与分离培养法检测NG时有12例结果不一致。世界卫生组织推荐培养法为用于诊断 NG 感染的“金标准”，通过感染者真阴性、真阳性的比较得出尿液快速法的敏感性和特异性分别为100.00 % 、92.50 % ，SAT法的敏感性和特异性分别为100.00 % 、97.50 %，SAT 法的特异性高于尿液快速法 5.50 %，见表2。

表 2 两种方法检测淋球菌的结果比较方法 阳性 阴性 真阳 真阴 敏感性 特异性尿液淋球菌快速法 52 148 40 160 100% 92.50%

NG-SAT 44 156 40 160 100% 97.50%

3 讨 论 淋病对人类生殖道感染的影响严重，其感染可增加其他性传播疾病感染的风险达5倍以上，广泛开展性病的早期筛查、早期诊断以及早期治疗极为重要 [4]。据调

查NG在不孕不育人群感染中增加趋势明显，且部分淋病患者因临床上无典型症状而被忽视，无症状淋病患者是传播淋病奈瑟菌感染的重要原因，慢性感染者还可导致不育。传统的NG检测方法存在检出率低、周期较长、特异性差等缺点，给诊断和治疗带来困难，建立高敏感、高特异、快速便捷的实验室诊断方法对临床诊断和治疗有重要意义[5]。本研究采用尿液快速检测法和SAT技术对不孕不育人群的尿液样本进行NG检测，以找到适合临床诊断的方法，并为其治疗提供合理的依据。尿液淋球菌快速检测法是实验室常用的方法之一, 其气泡的产生与淋球菌数量

的多少有关, 隐性感染者泡沫产生速度相对较慢，此项试验检测方法取材方便、操作简单、经济方便、患者容易接受，也便于快速诊断。但快速法敏感性强，易造成假阳性结果，SAT技术作为新一代的核酸扩增检测技术现已被广泛应用到病原体检测领域，该技术具有高灵敏性、高特异性、低污染、反应稳定等优点[6] ，其将实时荧光检测和核酸恒温扩增相结合，特异性提取尿液或拭子样本中的病原体 RNA；RNA在环境中容易降解，交叉污染少，降低了假阳性问题，有助于临床疗效监测；SAT采用磁珠法对特异性靶标进行捕获扩增检测，水相洗涤，有效减少假阴性，尤其是当患者正处于感染初期，SAT技术更能显示出其高敏感性。SAT法检测的是活菌中的RNA片段,阳性可提示病原体处于活性状态，对诊断有重要意义；患者经治疗后病菌死亡、临床症状消失，RNA在死亡的病原体中降解快速，有利于临床用药后的疗效观察及判愈。有研究表明，NG -SAT在尿液和拭子检测中的结果无差异，标本符合率高达 98.50 %，首段尿标本与拭子的敏感性相当[7]，由于部分无症状患者对生殖道检查取样的排斥，做非侵入性的尿液样本检测更易于采集接受，使用尿液作检测样本，避免拭子取样的尴尬和痛苦，提高了患者取样的依从性，减少了医生的工作量，简化了收集流程，具有较大的临床应用价值[8]。 笔者通过 100例有疑似症状的标本和 100例无明显症状的标本分别进行尿液淋球菌快速检测和 SAT检测，结果显示，尿液淋球菌快速检测法阳性率为为 26.00 ％，其中疑似标本阳性率占 44.00 %，无症状标本阳性率占 8.00 %；NG -SAT阳性率为

22.00 ％，其中疑似标本阳性率占 39.00%，无症状标本阳性率占 5.00%，SAT法与国内文献结果报道的 NG阳性率更相近，SAT法低于尿液快速检测法阳性率 4.00

%，该方法检测的假阳性率明显低于快速法。结果显示 100例无症状者的阳性率并不是很高，但仍有一定的隐性感染情况，不容忽视。研究表明该两种方法的符合率均高于 90.00％，其中快速检测法的阳性率最高，这可能是由于快速法的原理是淋球菌具有一种特异性酶与试剂发生生化反应并产生气泡。 正常情况下分泌物或尿中的杂菌的酶活性很低不致产生阳性反应，但当杂菌数量大大增加时可引起假阳性反应。同时严重蛋白尿和血尿也可影响结果，特别是女性分泌物标本，增多时会干扰试验产生假阳性[9]。结果显示，SAT 法检测疑似淋球菌感染患者尿液标本具有较高的灵敏度和特异度，通过 SAT法与尿液快速法检测 NG的结果比较，应用 SAT

法可以帮助辨别快速法造成的假阳性,对临床症状不典型和已治疗但未完全清除的患者，标本中含量不高的病原微生物，SAT法可以更好的检测出，避免了漏检，对于疑似病例的早期诊断具有重要临床意义[10]。 综上所述，SAT 技术既具备采样方便、耗时短、低污染的实验要求，又有敏感性高,特异性好，反应稳定，结果准确可靠等优点，还可用于用疗效观察和判愈评估，并为淋球菌的实验室诊断提供新的采样方式和检测方法，该技术更具有临床实用性，优于目前其他检测方法[11]。参考文献 [1] 肖秀美,高爽,杨旭,等. 2998例男性患者解脲脲原体、淋病奈瑟菌和沙眼衣原体感染调查分析[J].中国男科学杂志,2016,30(1):21-24.

[2] 邓冲.分离培养法与涂片镜检法在诊断女性淋球菌感染的临床应用比较[J].国际检验医学杂志,2013,34(14):1853-1854.

[3] 唐盛,林恒先,龚海涛,等.免疫荧光法检测奈瑟氏淋球菌方法的建立及其临床应用评价[J].标记免疫分析与临床,2011,18(2):107-111.

[4] 许丽琴 .术前白带常规和淋球菌检测在计划生育术中的意义分析 [J].当代医学,2011,17(23):84-85.

[5] 周志光, 胡永葵,黄霖庆.淋球菌快速检测法在高危场所人群现场调查中的应用[J].

临床和实验医学杂志,2010,9(20):1566-1567.

[6] 张津萍,龚匡隆,尤永燕,等.实时荧光核酸恒温扩增法检测泌尿生殖道淋球菌感染[J].国际皮肤性病学杂志,2010,36(3):137-139.

[7] 周璐,蔡筠,谢建生.实时荧光核酸恒温扩增技术在不孕育龄妇女病原体检测中的应用研究[J].医药前沿,2013,3(21):55-56.

[8] 马萍 ,聂庆东 ,殷敏 .3种方法检测淋病奈瑟菌的比较分析及意义 [J].医学检验,2012,2(9):126-128.

[9] 钟春燕,章小花,裘新民,等.实时荧光核酸恒温扩增技术在淋球菌检测中的应用评价[J].中国卫生检验杂志,2015,25(23):4041-4045.

[10] 郑雅萍,袁青,陈伟.实时核酸恒温扩增技术在奈瑟菌感染检测中的应用[J].浙江实用医学,2015,20(1):56-58.

[11] 高志华,金印,陈峰.实时荧光核酸恒温扩增技术检测尿液中淋球菌的分析[J].国际检验医学杂志,2012,33(4):463-464.

类别：临床实验室生物安全管理及分子诊断技术与临床应用903075

Gene XpertMTB/RIF 快速检测结核分枝杆菌及利福平的耐药性分析王伟 杨晶 王珂 李铮河南省胸科医院

项目基金：河南省医学科技攻关项目（201402031）[摘要 ] 目的 探讨利福平耐药实时荧光测量 PCR 结核分枝杆菌 /利福平技术（GeneXpert MTB/RIF）对结核分枝杆菌及对利福平的耐药性检测价值。方法 选取我院 2015年 9月—2017年 10月疑似肺结核患者 186例，进行 GeneXpert MTB/R

IF法快速检测、比例法药敏试验、改良罗氏固体培养、显微镜检查。结果 以改良罗 氏固体培养法检测结果作金标准， GeneXpert MTB/RIF 检测敏感度为90.91%（ 80/88）、特异度为 89.80%（ 88/98），两者具有较高一致性（K=0.806）；以痰涂片染色法检测结果作金标准，GeneXpert MTB/RIF检测敏感度为 87.38%（90/103）、特异度为 100.00%（83/83），两者具有较高一致性（K=0.861）；以比例法药敏试验结果作金标准，GeneXpert MTB/RIF检测利福平耐药敏感度为 90.91%（10/11）、特异度为 98.51%（66/67），两者具有较高一致性（K=0.894）。结论 采取 GeneXpert MTB/RIF法快速检测结核分枝杆菌可提高诊断准确度及特异度，且可准确检测利福平耐药性，为临床制定、调整治疗方案提供一定参考依据，选取耐药性较好药物进行治疗，对改善临床疗效具有重要作用。[关键词] GeneXpert MTB/RIF法；快速检测；结核分枝杆菌；利福平；耐药性Rapid detection of Mycobacterium Tuberculosis by GeneXpert MTB/RIF Method

and Analysis of its Resistance to Rifampin

[Abstract] Objective To investigate the detection value of the rifampin resistance real-

time fluorescence detection of PCR Mycobacterium tuberculosis / rifampin

（ GeneXpert MTB/RIF） in the drug resistance of mycobacterium tuberculosis to

rifampin. Methods A total of 186 cases of suspected tuberculosis patients from

September 2015 to October 2017 in our hospital were selected and rapidly detected by

GeneXpert MTB/RIF, proportional method of drug sensitivity test, improved Roche solid

culture and microscopic examination. Results The improved Roche solid culture method

was used as the gold standard; the sensitivity of GeneXpert MTB/RIF detection was

90.91% (80/88) and the specificity was 89.80% (88/98), which had high

consistency（K=0.806）. The test results of sputum smear staining was used as the gold

standard; the sensitivity of GeneXpert MTB/RIF detection was 87.38% (90/103) and the

specificity was 100% (83/83), which had high consistency（K=0.861）. The test results

of proportional method of drug sensitivity test was used as the gold standard; the

sensitivity of rifampin resistant of GeneXpert MTB/RIF detection was 90.91%(10/11) ,

the specificity was 98.51% (66/67), which had high consistency （ K=0.894 ） .

Conclusion The rapid detection of mycobacterium tuberculosis by GeneXpert MTB/RIF

can improve the accuracy and specificity of diagnosis. And it can accurately detect the

drug resistance of rifampicin. It provides a reference for clinical formulation and

adjustment of the treatment scheme, and the selection of high resistant drugs for

treatment is of great importance to the improvement of clinical efficacy.

[Keywords] GeneXpert MTB/RIF, Rapid detection, Mycobacterium tuberculosis,

Rifampin, Drug resistance

结核病为感染结核分枝杆菌（MTB）所致传染性病变多发类型，为全球范围内单因素致死率极高的一种疾病[1]。目前，临床多通过实验室检查对结核病予以诊断，当前我国主要采取传统抗酸染色法及培养法对结核病予以诊断，但其特异度及敏感度较低，且用时较长，易致使患者错失最佳治疗时机 [2-3]。因此，寻找一种准确度高、用时少的结核病和耐药性诊断措施成为研究热点，且对控制我国结核病严峻形势具有重要作用。近些年，GeneXpert MTB/RIF法应用价值得到普遍重视，其主要利用全自动半巢式实时 PCR 技术，选取 rpoB基因作靶基因，且能于 2 h内对结核分枝杆菌及利福平耐药性予以同时检测，可缩短检测用时 [4-5]。本研究选取我院疑似肺结核患者 186例，探讨GeneXpert MTB/RIF法应用价值。如下报告。1 资料与方法

1.1 一般资料 选取我院 2015年 9月—2017年 10月疑似肺结核患者 186例，其中男 127例，女 59例；年龄 22～81岁，平均（50.79±14.28）岁。所有受检者均知晓本研究，签署同意书，且本研究经我院伦理委员会审核同意。1.2 材料及方法1.2.1 材料 取所有受检者晨起时痰液标本，选用 GeneXpert MTB/RIF

Kit（Cepheid，USA，批号：05903）、MPT64胶体金免疫结核分枝杆菌鉴别检测试纸条、改良罗氏固体培养基、萋-尼式抗酸染液、SIRE药敏培养基、SR样品处理液 、标准菌 株为中国药品生物制品检定所提供的结核分枝杆菌H37Rv（ATCC27294）。1.2.2 方法 （1）结核分枝杆菌鉴定：经改良罗氏培养基所获取分枝杆菌参照胶体金免疫层析结核分枝杆菌鉴别试剂盒说明书进行鉴别，辨别非结核分枝杆菌及结核分枝杆菌；（2）GeneXpert MTB/RIF检测：取痰标本 1 ml置于前处理管（带有螺旋盖），优先选择痰标本内脓性及黏稠部分，于前处理管内加入 2 倍痰标本体积的处理液，拧紧螺旋盖，于生物安全柜中把前处理管放在涡旋振荡器上进行 10 s涡旋振荡，至块状痰标本不可见，室温条件下静置前处理管 15 min，充分液化痰标本，静置 10 min，若痰标本中存在未液化细小块状痰标本，则再次进行震荡，开启 Cartridge反应盒，以无菌吸管抽取处理后样本 2 ml置入 Cartridge反应盒，放于GeneGene Xpert检测系统实施全自动检测；（3）药敏试验：把液体培养呈阳性，且经抗酸染色镜检查确认是抗酸杆菌的标本进行比例法药敏试验。所有受检者各取同一耐药基因片段进行上述实验；（4）对利福平耐药试验：其测定基础为MTB

特异性分子信标早期 Ct值与晚期 Ct值之差（ΔCt值），若 ΔCt值＞3.5 则对利福平耐药，否则为对利福平敏感。1.3 统计学分析 通过 SPSS20.0对数据进行分析，计数资料 n（%）表示，χ2检验，GeneXpert MTB/RIF检测结果与检测金标准实施一致性（Kappa）检验（K＞0.750

为一致性较好），P＜0.05表示差异有统计学意义。

2 结果2.1 GeneXpert MTB/RIF检测和改良罗氏固体培养法比较 本组 186份痰标本进行检测，GeneXpert MTB/RIF检测阳性 90份，阳性率为 48.38%（90/186）；改良罗氏固体培养法检测阳性 88份，阳性率为 47.31%（88/186）；以改良罗氏固体培养法检测结果作金标准，GeneXpert MTB/RIF检测敏感度为 90.91%（80/88）、特异度为 89.80%（88/98），两者具有较高一致性（K=0.806）。见表 1。

表 1 GeneXpert MTB/RIF检测和改良罗氏固体培养法比较（n=186）GeneXpert MTB/RIF

改良罗氏固体培养法 总计阳性 阴性阳性 80 10 90

阴性 8 88 96

总计 88 98 186

2.2 GeneXpert MTB/RIF检测和痰涂片染色法检测比较 痰涂片染色法检测阳性103 份，阳性率为 55.38%（103/186）；以痰涂片染色法检测结果作金标准，GeneXpert MTB/RIF 检 测 敏 感 度 为 87.38% （ 90/103 ） 、 特 异 度 为100.00%（83/83），两者具有较高一致性（K=0.861）。见表 2。

表 2 GeneXpert MTB/RIF检测和痰涂片染色法检测比较（n=186）GeneXpert MTB/RIF

痰涂片染色法 总计阳性 阴性阳性 90 0 90

阴性 13 83 96

总计 103 83 186

2.3 GeneXpert MTB/RIF检测对痰标本内利福平耐药性检测分析 本组 186例痰培养阳性 87例，其中无GeneXpert MTB/RIF检测结果者 9例，最终可实施GeneXpert 

MTB/RIF对比分析者共 78例，以比例法药敏试验结果作金标准，GeneXpert MTB/

RIF检测利福平耐药敏感度为 90.91%（10/11）、特异度为 98.51%（66/67），两者具有较高一致性（K=0.894）。见表 3。表 3 GeneXpert MTB/RIF检测对痰标本内利福平耐药性检测分析（n=78）

GeneXpert MTB/RIF比例法药敏试验 总计耐药 敏感

耐药 10 1 11

敏感 1 66 67

总计 11 67 78

3 讨论我国为结核病高发地区，及早确诊疾病并采取有效治疗措施对改善患者临床疗

效及预后极为重要。目前，临床多采取涂片镜检（金胺“O”荧光染色及糠酸染色）及MTB培养法对结核病予以诊断，前者操作简单、迅速，但检出率较低，特别是对无菌标本（如脑脊液等）予以检测时极易造成漏诊；而后者为结核病诊断金标准，但比例法试验及罗氏培养法检测结果通常需 8周后才可检出，而药敏试验及MGIT液体培养虽在一定程度缩短了检测时间，但仍需 4周左右，易导致患者错失最佳治疗时机[6]。

GeneXpert MTB/RIF法为近些年在结核病临床诊断中得到推广应用的措施，相关研究指出，GeneXpert MTB/RIF法采取多个光谱光学检测可实时定量提供较为准确的 PCR结果，避免误诊或漏诊，且该技术应用独立的自成体系一次性反应盒，可提高设备安全性[7]。同时，GeneXpert MTB/RIF法操作较简单，其经系统软件对PCR予以实时监测，可于 PCR 完成后自动给出试验结果，且其配置诸多模块同时进行多个检测，其中任意一个反应均可随时终止或启动，灵活性较高。另有学者指出，GeneXpert MTB/RIF 技术将定量检测、样品准备及扩增进行统一，且其具备极速制冷与加热能力，可于 2 h内提供稳定、准确试验结果，可有效弥补传统诊断方式用时较长这一不足，利于患者及早接受有效治疗[8]。我国相关学者证实，同时采

取GeneXpert MTB/RIF、固体培养法、抗酸染色法对结核患者予以检测，结果表明GeneXpert MTB/RIF敏感度与特异度均较高 [9]。而本研究中，GeneXpert MTB/RIF

检测与改良罗氏固体培养法、痰涂片染色法检测结果具有较好一致性，且其对利福平耐药敏感度及特异度较高，表明 GeneXpert MTB/RIF在抗酸染色阳性痰标本结核分枝杆菌及利福平耐药性检测中具有较高应用价值，且其检测用时较短，利于临床迅速制定有效治疗方案。此外，张燕等[10]指出，痰标本量不足或标本不合格及所感染分枝杆菌非分枝杆菌复合群均会对 GeneXpert MTB/RIF检测结果造成一定影响，因此临床实际应于诊断过程中严格把控痰标本质量，避免因痰标本问题造成漏诊或误诊，致使患者延误最佳治疗时机综上所述，采取 Gene Xpert MTB/RIF法快速检测结核分枝杆菌可提高诊断准

确度及特异度，且可准确检测利福平耐药性，为临床制定、调整治疗方案提供一定参考依据，选取耐药性较好药物进行治疗，对改善临床疗效具有重要作用。参考文献[1] 肖慎荣,周桂莲.缩宫素联合心得安地西泮治疗肺结核咯血对照观察[J].临床心身疾病杂志,2011,17(6):569-569.

[2] 牛海军,王歌,李明虎.GeneXpert MTB/RIF检测在肺结核诊断中的价值评估[J].中国防痨杂志,2017,39(8):829-832.

[3] 刘爱梅,苏玉,张琪,等.GeneXpert MTB/RIF试验在肺结核诊断中的应用评价[J].中国医师进修杂志,2017,40(6):500-504.

[4] Zeka A N,Tasbakan S,Cavusoglu C.Evaluation of the GeneXpert MTB/RIF Assay

for Rapid Diagnosis of Tuberculosis and Detection of Rifampin Resistance in Pulmonary

and Extrapulmonary Specimens[J].J Clin Microbiol,2011,49(12):4138-4141.

[5] 伊惠霞,侯新月,王泉,等.Xpert MTB/RIF系统快速检测结核分枝杆菌及利福平耐药性研究[J].新疆医科大学学报.2014,(12):1643-1646.

[6] Ioannidis P,Papaventsis D,Karabela S,et al.Cepheid GeneXpert MTB/RIF assay for

Mycobacterium tuberculosis detection and rifampin resistance identification in patients

with substantial clinical indications of tuberculosis and smear-negative microscopy

results[J].J Clin Microbiol,2011,49(8):3068-3070.

[7] 陈子芳,孙本海,霍丽丽,等.GeneXpert MTB/RIF 技术在结核性胸膜炎诊断及利福平耐药检测中的价值[J].中国卫生检验杂志.2017,(6):819-821.

[8] 赵金云,金法祥,许文芳,等. 应用 GeneXpertMTB/RIF对耐多药MTB及 RIF耐药性的快速检测[J].中华医院感染学杂志,2017,27(19):4340-4343.

[9] 周洪经,郭明日,冯爽,等.Xpert MTB/RIF在快速诊断肺结核及利福平耐药中的临床应用[J].国际检验医学杂志.2016,(18):2568-2570.

[10] 张燕,李波.GeneXpert MTB/RIF 快速检测法结核分支菌及其对利福平耐药性的临床价值评估[J].现代实用医学.2016,(10):1361-1362.