【作者】 高敏；

【导师】 童先宏；

【作者基本信息】 安徽医科大学 ， 临床检验诊断学， 2021， 硕士

【摘要】 目的目前缺乏合适的临床标本来诊断复发性流产(recurrent spontaneous abortion,RSA)。本研究通过流式检测正常人和复发性流产患者月经血NK(menstrual blood natural killer cells,MBNK)、蜕膜NK(decidual natural killer cells,d NK)、外周血NK(peripheral blood natural killer cells,PBNK)细胞及其相关亚群的表达情况,将经血结果与蜕膜组织和外周血结果进行比较,探索经血标本对预测复发性流产发生可行性。方法转录组学首先分析证明正常月经血NK与正常蜕膜NK相似,接下来使用复发性流产患者月经血NK对子宫内膜NK(endometrium NK cells,u NK)状态进行评估,具体方法为蜕膜标本收集:收集2019年9月-2020年7月于中国科学技术大学附属第一医院妇产科人工流产手术室行无痛人流的患者蜕膜组织共25份,其中正常对照组蜕膜取自自愿选择终止妊娠的健康妇女(n=15),实验组蜕膜取自经历两次及以上胚胎停止发育史或自然流产史且胚胎及夫妻双方染色体核型均正常的妇女(n=10),这两组人群均为早期妊娠。经血标本采集:于月经周期第二天采集经历正常生育史且平素月经规律的健康女性月经血作为对照组标本(n=10),有两次及以上复发性流产史且染色体核型正常的女性月经血作为实验组标本(n=25),在采集经血的同时采集外周血标本。所有受试者都没有染色体异常(自己或丈夫)、子宫异常、自身免疫性疾病或其他影响激素平衡的药物治疗等风险因素。将获得的所有样本均进行流式检测分析,主要检测NK(natural killer cells)细胞表面标志分子CD56,CD16;组织驻留分子CD49a,EOMES;抑制性受体CD158a,CD158b,GITR;活化性受体NKP30,NKP44,NKG2D。观察比较上述分子在正常组和实验组的三种样本中的表达差异,通过受试者工作特征(receiver operating characteristic,ROC)曲线来计算差异分子对RSA的预测效能。结果(1)基因转录组学证明正常人月经血NK与蜕膜NK具有相似性,与外周血NK有区别。(2)流式检测发现与对照组相比,流产组的蜕膜和经血中CD3~+T细胞比例增加,CD56~+NK细胞比例降低,而外周血中结果相反。(3)与对照组相比,流产组蜕膜及经血中CD56~+CD16~+表达明显升高,CD56~+CD49a~+表达降低,外周血中CD56~+CD16~+表达升高,CD56+CD49a+表达无明显差异。(4)与对照组相比,流产组蜕膜及月经血中CD49a~+CD16~-、CD49a~+EOMES~+NK细胞表达减少,CD49a~-CD16~+NK细胞表达增加,外周血中两组间无明显差异;(5)与对照组相比,流产组蜕膜、经血NK细胞抑制性受体CD158a表达降低,在外周血中表达无显著性差异;抑制性分子CD158b在经血和外周血中表达降低,而在蜕膜中表达无显著性差异;GITR在正常人与流产组的蜕膜、经血与外周血中表达均无显著性差异。(6)与对照组相比,活化性受体NKP30在流产组的蜕膜、经血与外周血中表达均无显著性差异;活化性受体NKP44在流产组的蜕膜、经血中表达均无显著性差异,在外周血中表达显著降低;NKG2D在流产组的蜕膜中表达均无显著性差异,在经血中表达显著升高,在外周血中表达显著降低。(7)经血NK细胞与外周NK细胞相比,对复发性流产的发生具有很高的预测效能。CD3~-CD56~+NK,CD56~+CD16~+NK,CD56~+CD49a~+NK,CD49a~+CD16~-NK,C D49a~+EOMES~+NK细胞子集对区分正常人和流产患者的曲线下面积(area under c urve,AUC)值均达到0.7以上。当使用CD49a~+EOMES~+NK,CD56~+CD49a~+NK,CD49a~+CD16~-NK进行预测时准确度均达到80%以上。结论(1)经血NK细胞与蜕膜NK具有相似性;(2)流产患者经血中存在NK细胞及其亚群表达异常,尤其表现在CD16与CD49a分子上,且与蜕膜NK表达情况具有很高的一致性;(3)月经血NK细胞亚群CD49a~+EOMES~+NK,CD56~+CD49a~+NK,CD49a~+CD16~-,CD56~+CD16~+可以用来诊断复发性流产,具有较高的诊断效能。更多还原

【Abstract】 ObjectiveThe expression of NK cells and NK cells related subsets in menstrual blood,decidua and peripheral blood of control group and recurrent spontaneous abortion(RSA)group were detected by flow cytometry,The results of menstrual blood,decidua and peripheral blood were compared to explore the feasibility of menstrual blood samples in predicting recurrent spontaneous abortion.MethodsTranscriptome analysis showed that NK cells in normal menstrual blood was similar to normal decidua NK(d NK)cells,then endometrium NK(u NK)cells status of RSA patients were evaluated by menstrual blood NK(MBNK)cells.Decidua samples(n=15)from normal pregnancies were obtained from women with selective pregnancy terminations,abnormal decidua samples(n=10)were obtained from women who experienced recurrent spontaneous abortion and had a normal karyotype.Menstrual blood(n=10)on the second day of the menstrual cycle was collected from healthy women who had normal reproductive history and regular menstruation,menstrual blood specimens(n=25)were obtained from patients who experienced recurrent spontaneous abortion and had a normal karyotype,meanwhile,peripheral blood was collected.All samples were collected from the First Affiliated Hospital of USTC.None of the subjects had risk factors such as chromosomal abnormalities(self or husband),uterine abnormalities,autoimmune diseases or other drug treatments that affected hormone balance.All samples obtained were analyzed by flow cytometry,mainly including NK cell surface marker molecules CD56 and CD16;Tissue resident molecule CD49a,EOMES;Inhibitory receptor CD158a,CD158b,GITR;Activated receptor NKP30,NKP44,NKG2D.The expression differences of the above molecules in the normal group and the experimental group were observed and compared.ROC curve was used to calculate the prediction efficiency of differential molecules for RSAResult(1)Transcriptomics proved that NK in normal menstrual blood was similar to decidua and different from peripheral blood.(2)Flow cytometry analysis showed the proportion of CD3~+T cells in decidual and menstrual blood of the RSA group was increased,CD56~+NK cells was decreased,while the results in peripheral blood were opposite.(3)Compared with control group,the expression of CD56~+CD16~+NK in decidua and menstrual blood of the RSA group was significantly increased,CD56~+CD49a~+NK was decreased;CD56~+CD16~+NK expression was increased in peripheral blood,and there was no different of CD56~+CD49a~+NK(4)Compared with control group,the expression of CD49a~+CD16~-and CD49a~+EOMES~+NK cells in decidua and menstrual blood of the RSA group were decreased,CD49a~-CD16~+NK cells were increased;there were no significant difference in peripheral blood.(5)Compared with control group,the inhibitory receptor CD158a in decidua NK and menstrual blood NK were decreased in the RSA group,there was no significant difference in peripheral blood NK(PBNK);CD158b was no significant difference in decidua and decreased in menstrual and peripheral blood;GITR was no significant difference of in decidua,menstrual blood and peripheral blood of RSA group.(6)Compared with control group,the activated receptor NKP30 was not significantly different in the decidua,menstrual blood and peripheral blood of the RSA group;NKP44was no significant difference in decidua and menstrual blood of the RSA group,decreased in peripheral blood;NKG2D in decidua of the RSA group was no significant difference,increased in menstrual blood and decreased in peripheral blood.(7)Menstrual blood NK cells have a high predictive effect on recurrent spontaneous abortion compared with peripheral NK cells.The AUC of CD3~-CD56~+NK,CD56~+CD16~+NK,CD56~+CD49a~+NK,CD49a~+CD16~-NK,CD49a~+EOMES~+NK cells subsets were greater than 0.7.The accuracy more than 80%when CD49a~+EOMES~+NK,CD56~+CD49a~+NK and CD49a~+CD16~-NK for prediction,Conclusion(1)Menstrual blood NK cells are similar to decidual NK cells;(2)Compared with the control group,the expression of NK cells and subsets in menstrual blood of patients with RSA was abnormal,which was highly consistent with decidua.(3)Menstrual blood NK cell subsets CD49a~+EOMES~+NK,CD56~+CD49a~+NK,CD49a~+CD16~-,and CD56~+CD16~+can diagnose recurrent spontaneous abortion with high diagnostic efficiency.更多还原