【作者】 胡敏；

【导师】 洪莉；

【作者基本信息】 武汉大学 ， 妇产科学， 2018， 博士

【摘要】 目的复发性流产(Recurrent Spontaneous Abortion,RSA)影响到世界上大约1%的生育能力的夫妇。虽然大量基础研究已经阐明了其可能的发生机制,但目前仍有约有一半的复发性流产的患者病因不明。由于其高发病率和低治愈率,许多患者及家庭遭受了很大的痛苦。在妊娠的的建立和维持中,滋养层细胞的增殖、迁移和侵袭扮演了重要角色。据报道,滋养层细胞功能受损与复发性流产有关,但潜在的机制仍然未知。CD44变体结构域6(CD44v6)是数十年来已知表达于胎盘的跨膜糖蛋白。本实验研究CD44v6、NFκB在复发性流产的表达及其相关性,并研究了 CD44v6-NFκB通路对滋养细胞增殖及侵袭力的影响,及其相关的作用机制,进一步阐明CD44v6对复发性流产的作用机制。方法收集2015年3月至2016年3月在武汉大学人民医院妇产科因稽留流产或计划外妊娠要求人工流产终止妊娠的患者的流产组织,进行如下分组:①实验组:具有连续两次或两次以上流产史患者,此次妊娠经B超确诊为胚胎停育,要求人工流产终止妊娠者10例,并排除以下情况:夫妻双方任何一方有染色体或基因方面缺陷或恶性肿瘤病史;母体有子宫结构异常,或有弓形虫、巨细胞病毒、风疹病毒、单纯疱疹病毒感染、梅毒等的感染、或有抗凝脂综合征、高血压、糖尿病、甲状腺功能异常等慢性疾病病史及药物治疗史的患者;②对照组:无不良孕产史且此次妊娠为正常妊娠要求人工流产终止妊娠的患者10例。免疫组化检测两组流产组织中CD44v6、NF κ B的表达水平。培养滋养细胞HTR-8/SVneo,并分为两组:①实验组:通过LipofectamineTM 2000向滋养细胞系HTR-8/SVneo细胞转染CD44v6特异性siRNA;②阴性对照组:通过LipofectamineTM 2000向滋养细胞系HTR-8/SVneo细胞转染空载体;③空白对照组:未转染脂质体的HTR-8/SVneo细胞。检测各组细胞转染后CD44v6、NFκB的表达水平,并行细胞克隆实验及Transwell细胞侵袭实验检测转染后各组滋养细胞增殖及侵袭功能,同时行免疫荧光实验确定各组细胞CD44v6、NF κ B的定位及定量情况。结果(1)免疫组织化学实验表明,与正常妊娠患者人工流产的绒毛组织相比,复发性流产患者流产组织中CD44v6的和NF-κ B的表达水平明显下降。(2)与阴性对照组及空白对照组相比,向滋养细胞HTR-8/SVneo转染CD44v6特异性siRNA可以显著降低CD44v6的mRNA水平,人滋养细胞CD44v6基因沉默表达模型成功建立。(3)与阴性对照组及空白对照组相比,向滋养细胞HTR-8/SVneo转染CD44v6特异性siRNA可以显著降低NFκB的mRNA水平(P<0.05);(4)细胞克隆实验结果表明,与阴性对照组及空白对照组相比,向滋养细胞HTR-8/SVneo转染CD44v6特异性siRNA后滋养细胞增殖能力明显下降(P<0.05);(5)Transwell细胞侵袭实验结果表明,与阴性对照组及空白对照组相比,向滋养细胞HTR-8/SVneo转染CD44v6特异性siRNA后滋养细胞侵袭能力明显下降(P<0.05);(6)免疫荧光染色结果表明,当CD44v6在滋养层HTR-8/SVneo中被特定的siRNA降调节时,NFκB的细胞核内聚集被显著削弱了。结论1、复发性流产患者滋养细胞CD44v6及NF κ B表达下降,CD44v6及NF κ B的异常调节与复发性流产相关;2、人滋养细胞中CD44v6表达水平的下降可引起NFκB表达的下降,人滋养细胞中存在CD44v6-NF κ B信号通路;3、人滋养细胞中CD44v6表达水平的下降可导致滋养细胞增殖、侵袭能力降低,并使及NFκB的细胞核聚集明显下降,提示CD44v6可能通过调控人滋养细胞NFκB表达及细胞核聚集水平,从而影响人滋养细胞的增殖及侵袭,最终导致复发性流产。更多还原

【Abstract】 ObjectiveRecurrent Spontaneous Abortion(RSA)affects about 1 percent of the world’s fertile couples.Although a number of basic studies have elucidated its possible mechanism,about half of the causes of patients with recurrent abortion are still unknown.Due to its high incidence and low cure rate,many patients and families suffered a lot.The proliferation,migration and invasion of trophoblast cells play an important role in the establishment and maintenance of pregnancy.Damage to trophoblast cells is reported to be associated with recurrent abortion,but the underlying mechanism remains unknown.CD44 variant domain 6(CD44v6)is a transmembrane glycoprotein known to be expressed in placenta for decades.In this study,the expression and correlation of CD44v6 and Nuclear Factor NF kappa B in recurrent abortion were studied,and the effects of CD44v6 and NF expressed B pathway on the proliferation and invasion of trophoblast cells were studied,as well as the related mechanism of action,further clarifying the mechanism of action of CD44v6 on recurrent spontaneous abortion.MethodsThe study was conducted from March 2015 to March 2016 in the outpatient operation room of the Department of Gynaecology and Obstetrics of Renmin Hospital at Wuhan University.Patients who had at least two consecutive spontaneous abortions and were 7-10 weeks pregnant were allocated to the RSA group(n = 10),and the women with no history of spontaneous abortion and also with a gestation period of 7～10 weeks were enrolled into the control group(n = 10).All participants were non-smokers,not on medication,and had regular menstrual cycles.The expression levels of CD44v6 and NF κ B in the two groups were detected by immunohistochemistry.The trophoblast cells HTR-8/SVneo were cultured and divided into two groups:(1)the experimental group:transfection of CD44v6 specific siRNA into trophoblast cell line HTR-8/SVneo cells via LipofectamineTM 2000;(2)the negative control group:transfected empty vector into trophoblast cell line HTR-8/SVneo by LipofectamineTM 2000;(3)the blank control group:HTR-8/SVneo cells without liposome transfection.The expression levels of CD44v6 and NF κ B were detected after transfection,and the proliferation and invasion functions of trophoblast cells were detected by cloning formation assay and Transwell cell invasion experiment,what’s more,and the localization and quantification of CD44v6 and NF κ B were determined by immunofluorescence experiment.Results1.In patients with RSA,the expression of CD44v6 and NFκB decreased,and the abnormal regulation of CD44v6 and NFκB was related to RSA.2.Downregulation of CD44v6 in human trophoblast cells can lead to the decrease of the expression level of NFκB.3.Downregulation of CD44v6 in human trophoblast cells can result in decreased proliferation and invasion ability of trophoblast cells,and significantly reduce the nuclear aggregation of NFκB.ConclusionsCD44v6 may affect the proliferation and invasion of human trophoblast cells by regulating the expression and nuclear aggregation of NF κ B in human trophoblast cells,and eventually lead to RSA.更多还原