【作者】 辛虹；

【导师】 王惠兰；

【作者基本信息】 河北医科大学 ， 外科学， 2011， 博士

【摘要】 子痫前期(preeclampsia,PE)是妊娠期特有的疾病,是妊娠期高血压疾病最常见的类型。流行病学调查显示其发病率在我国约为9.4%-10.4%,国外报道其发病率为7%-12%。尽管围产保健技术不断进步,该病仍严重影响母儿健康,是导致孕产妇和围生儿病率及死亡率增加的主要原因之一。尽管国内外学者对其病因及发病机制做了大量的研究,目前子痫前期的发病机制不明确,尚未发现明确单一致病因素,系多因素综合作用所致。膜联蛋白(annexins)是一种非常古老而又广泛存在的蛋白,是一种以钙离子依赖性方式与酸性磷脂结合的蛋白质。大量的研究显示,膜联蛋白在胞吞、胞吐作用、细胞间以及细胞与细胞外基质间的相互作用、离子通道的形成等多种生理过程中起作用。研究发现,某些疾病的发生与膜联蛋白家族在细胞中的表达密切相关。在annexin家族中,annexin A2和annexin A5与凝血-纤溶密切相关。长期以来,研究发现annexin A2和annexin A5与不良妊娠结局如复发性流产、抗磷脂综合症的胎盘变化密切相关,但其与子痫前期的关系尚缺乏深入研究。第一部分缺氧条件下滋养细胞的生长及胎盘滋养细胞凋亡和病理检测目的:研究体内外缺氧环境对滋养细胞在生长、凋亡的影响及对胎盘的影响。方法:采用缺氧培养技术检测体外缺氧情况下滋养细胞的生长。通过流式细胞术检测体内缺氧情况下(子痫前期患者体内的缺氧状态)滋养细胞的凋亡。同时通过HE染色检测子痫前期患者胎盘组织的病理变化。结果:1体外滋养细胞缺氧状态培养发现,随着缺氧时间的延长,滋养细胞生长受限,膜联蛋白表达水平下调。2流式细胞术检测发现,子痫前期(体内缺氧情况下),滋养细胞凋亡尤其是早期凋亡发生改变:早发型轻度子痫前期组为3.21%,早发型子痫重度前期组为5.32%,晚发型轻度子痫前期组为2.43%,晚发型重度子痫前期组为4.28%,正常妊娠组为1.05%。3在正常妊娠者的胎盘组织中未发现血管内微血栓的形成。而在子痫前期患者胎盘组织中可以见到胎盘血管内微血栓的形成,且随着病情程度的加重,微血栓形成逐渐增多。结论:缺氧情况下,体外滋养细胞生长受限;体内滋养细胞(胎盘滋养细胞)凋亡尤其是早期凋亡明显增加。同时,子痫前期患者胎盘组织血管中可见微血栓形成。第二部分膜联蛋白A5在子痫前期患者体内的表达目的:探讨膜联蛋白A5(annexinA5)在子痫前期及正常妊娠孕妇外周血及胎盘组织中的表达及其与子痫前期发病的关系。方法:选择2007年12月-2009年12月在河北医科大学第二医院住院分娩患者早发型子痫前期(轻度、重度)、晚发型子痫前期(轻度、重度)及健康妊娠孕妇各30例,共150例,收集其外周血及胎盘组织,应用免疫组化研究annexin A5在胎盘组织中的定位、应用Western blot、RT-PCR测定患者外周血及胎盘组织中annexin A5的表达。结果:1通过免疫组化检测发现,膜联蛋白A5(annexin A5)主要在胎盘滋养细胞表面高表达。与正常妊娠相比,子痫前期患者胎盘滋养细胞表面annexin A5染色减少。2通过Western blot检测子痫前期患者胎盘组织及外周血中annexin A5的表达水平。胎盘组织及外周血中annexin A5水平:早发型轻度子痫前期组分别为0.54±0.12及0.62±0.17,早发型重度子痫前期组分别为0.47±0.15及0.56±0.24,晚发型轻度子痫前期组分别为0.74±0.23及1.08±0.32,晚发型重度子痫前期组分别为0.68±0.28及0.72±0.21,正常妊娠组分别为1.73±0.35及1.55±0.27。与正常妊娠相比,子痫前期患者组织中的膜联蛋白A5(annexin A5)表达明显减少。3通过RT-PCR技术检测胎盘组织中annexin A5 mRNA的表达水平:早发型轻度子痫前期组为25.0±3.04,早发型重度子痫前期组为24.8±2.96,晚发型轻度子痫前期组为25.4±3.92,晚发型重度子痫前期组为25.1±2.74,正常妊娠组为30.6±2.68。结论:通过免疫组化和Western blot检测发现,膜联蛋白A5(annexin A5)主要在胎盘滋养细胞表面高表达,与正常妊娠相比,子痫前期患者胎盘滋养细胞annexin A5表达下调,可能影响胎盘凝血-纤溶系统,促使子痫前期患者高凝状态的形成,与子痫前期的发生、发展相关。第三部分膜联蛋白A2在子痫前期患者体内的表达目的:探讨膜联蛋白A2(annexin A2)在子痫前期及正常妊娠孕妇外周血及胎盘组织中的表达及其与子痫前期发病的关系。方法:选择2007年12月-2009年12月在河北医科大学第二医院住院分娩患者早发型子痫前期(轻度、重度)、晚发型子痫前期(轻度、重度)及健康妊娠组患者各30例,共150例,收集其外周血及胎盘组织,应用免疫组化研究annexin A2在胎盘组织中的定位、应用Western blot、RT-PCR测定患者外周血及胎盘组织中annexin A2的表达。结果:1通过免疫组化发现,膜联蛋白A2(Annexin A2)主要在胎盘滋养细胞表面高表达。与正常妊娠相比,子痫前期患者胎盘滋养细胞表面annexin A2染色减少。2通过Western blot检测子痫前期患者胎盘组织及外周血中annexin A2的表达水平。胎盘组织及外周血中annexin A2水平:早发型轻度子痫前期组分别为0.77±0.15及0.63±0.09,早发型重度子痫前期组分别为0.76±0.19及0.66±0.13,晚发型轻度子痫前期组分别为0.74±0.23及0.71±0.23,晚发型重度子痫前期组分别为0.81±0.35及0.59±0.14,正常妊娠组分别为1.82±0.18及0.50±0.04。与正常妊娠相比,子痫前期患者组织中的膜联蛋白A2(annexin A2)表达明显减少。3通过RT-PCR技术检测胎盘组织中annexin A2 mRNA的表达水平:早发型轻度子痫前期组为27.94±1.49,早发型重度子痫前期组为27.23±1.26,晚发型轻度子痫前期组为28.27±0.85,晚发型重度子痫前期组为28.54±0.65,正常妊娠组为32.34±0.71。结论:通过免疫组化和Western blot发现,膜联蛋白A2(annexin A2)主要在胎盘滋养细胞表面高表达,与正常妊娠相比,子痫前期患者胎盘滋养细胞annexin A2表达下调,抑制了子痫前期患者的纤溶系统,促使子痫前期患者体内高凝状态的形成,与子痫前期的发生、发展相关。第四部分胎盘组织膜联蛋白A5提取纯化和体外抗凝试验目的:探讨体内及体外膜联蛋白A5(annexin A5)的抗凝作用及其对体内凝血系统的影响。方法:选择2007年12月-2009年12月在河北医科大学第二医院住院分娩健康妊娠者,收集其胎盘组织进行annexin A5提纯。通过体外实验测定不同浓度annexin A5对凝血系统的影响。同时收集子痫前期组及正常妊娠组患者的外周血,测定凝血功能。结果:1.当annexin A5的浓度≥30μg/ml时,APTT逐渐延长,各组间有统计学差异并呈梯度性依赖性增加。2.与正常妊娠组比较,子痫前期患者外周凝血功能检测未见明显差异,但D-Dimmer升高。结论:Annexin A5的抗凝活性同其浓度密切相关。当annexin A5浓度≥30μg/ml,APTT与annexin A5浓度呈依赖性相关。随着annexin A5浓度增加,APTT逐渐延长。子痫前期患者外周血的APTT未见明显变化,但D-Dimmer升高。第五部分相关抗体的检测及分析目的:检测子痫前期及正常妊娠孕妇外周血中的相关抗体,并分析其在子痫前期发病机制中的作用。方法:应用ELISA方法检测子痫前期及正常妊娠孕妇外周血中的anti-annexin A5、anti-annexin A2、anti-β2GPI、ACL水平。结果:1与正常妊娠相比,子痫前期患者外周血中anti-annexin A5水平增加,但无统计学意义(P=0.83)。2与正常妊娠相比,子痫前期患者外周血中anti-annexin A2水平增加,且具有显著性差异(P=0.02)。但子痫前期各组之间,早发型子痫前期高于晚发型子痫前期,重度子痫前期高于轻度子痫前期,但各组之间无统计学差异(P=0.69)。3与正常妊娠相比,子痫前期患者外周血中anti-β2GPI水平增加,且具有显著性差异(P=0.04)。但子痫前期各组之间,早发型子痫前期高于晚发型子痫前期,重度子痫前期高于轻度子痫前期,但各组之间无统计学差异(P=0.79)。4与正常妊娠相比,子痫前期患者外周血中ACL阳性率增加。结论:子痫前期患者外周血中的抗体可与相应抗原结合,从而影响了annexin A2、annexin A5的功能,破坏了体内的凝血-纤溶系统平衡,促使子痫前期患者体内高凝状态的形成。更多还原

【Abstract】 Preeclampsia is a disease which occures only in pregnancy. Epidemiological survey of its incidence in China is about 9.4% -10.4%, and foreign reports are 7% -12%. Although a lot of researches were done on the pathogenesis of PE, the mechanism remains largely unknown. And it cause maternal and child morbidity and mortality.Annexins is a very ancient and widespread protein, which is a calcium-dependent manner to acidic phospholipid binding proteins. A large number of studies have shown that annexins play a role in a variety of physiological processes, including endocytosis, exocytosis, cells and extracellular matrix interactions, formation of ion channels. Some diseases is associated with the expression and the activity of annexins. In the annexin family, annexin A2 and annexin A5 are mainly related to the coagulation and fibrinolysis systerm. For a long time, the study shows that annexin A2 and annexin A5 are closely related to the adverse pregnancy outcomes, such as recurrent miscarriage, antiphospholipid syndrome. But the relationship of PE and annexins are rare. Part one The growth and apoptosis of trophoblast cells in hypoxia and pathological test.Objective: To investigate the growth and apoptosis of trophoblast cells in hypoxia.Methods: Detect the growth of trophoblast cells in hypoxia in vitro. Trophoblast apoptosis in hypoxic in vivo were detceted by flow cytometry. Also placental pathology was detected by immunohistochemistry.Results:1 Trophoblast cells in hypoxia in vitro was found that with prolonged hypoxia, trophoblast growth restriction, decreased protein levels.2 In hypoxia, the apoptosis of trophoblast cell, especially early apoptosis increased: early-onset mild preeclampsia group was 3.21%, early-onset severe preeclampsia group was 5.32%, late-onset mild preeclampsia group was 2.43%, late-onset severe preeclampsia group was 4.28%, and the healthy pregnant group was 1.05%.3 Compared with normal pregnancy, the formation of microthrombi can be seen in placenta of preeclampsia with disease severity.Conclusion: In hypoxia, the JAR cell growth was restriction in vitro, the apoptosis of trophoblastic in vivo (placental trophoblast cells) was significantly increased, especially the early apoptosis. And microthrombosis was found in the placenta of PE. Part two The expression of annexin A5 in patients of preeclampsiaObjective: To investigate the annexinA5 in maternal blood and placental tissue and its relationship with the pathogenesis of preeclampsia.Methods: 120 preeclampsia and 30 matched normal pregnant controls were included in the study. Expression of annexin A5 mRNA in placental tissues was analyzed using quantitative real time RT-PCR. Annexin A5 protein in placentas as well as blood was determined by western blot analysis and immunohistochemistry.Results: The expression levels of annexin A5 mRNA were significantly decreased in preeclampsia compared to normal pregnancies. The levels of annexin A5 protein in placentas were also significantly reduced in patients with preeclampsia, compared with healthy pregnant women.1 Annexin A5 mainly expressed in the surface of trophoblast cell. Compared with normal pregnancy, the expression of annexin A5 in placental trophoblast of preeclampsia cell surface is reduced.2 The expression level of annexin A5 in placental tissue and peripheral blood. Placental tissue and peripheral blood levels of annexin A5: early-onset mild preeclampsia group are 0.54±0.12 and 0.62±0.17, early-onset severe preeclampsia group are 0.47±0.15 and 0.56±0.24, late-onset mild preeclampsia group are 0.74±0.23 and 1.08±0.32, late-onset severe preeclampsia group are 0.68±0.28 and 0.72±0.21, and healthy pregnant group are 1.73±0.35 and 1.55±0.27.3 The mRNA levels of annexin A5 by RT-PCR in placental tissue: early-onset mild preeclampsia group is 25.0±3.04, early-onset severe preeclampsia group is 24.8±2.96, late-onset mild preeclampsia group is 25.4±3.92, late-onset severe preeclampsia group is 25.1±2.74, and healthy pregnancy group is 30.6±2.68.Conclusion: Annexin A5 mainly expresses on the surface of trophoblast cell. Compared with normal pregnancy, annexin A5 in placental of preeclampsia was decreased, which affect placental blood coagulation - fibrinolytic system. It may play an important role in the occurrence and the development of preeclampsia. Part three The expression of annexin A2 in patients of preeclampsiaObjective: To investigate the annexinA2 in maternal blood and placental tissue and its relationship with the pathogenesis of preeclampsia.Methods: 120 preeclampsia and 30 matched normal pregnant controls were included in the study. Expression of annexin A2 mRNA in placental tissues was analyzed using quantitative real time RT-PCR. Annexin A2 protein in placentas as well as blood was determined by western blot analysis and immunohistochemistry.Results: The expression levels of annexin A2 mRNA were significantly decreased in preeclampsia compared to normal pregnancies. The levels of annexin A2 protein in placentas were also significantly reduced in patients with preeclampsia, compared with healthy pregnant women.1 Annexin A2 mainly expressed in the surface of trophoblast cell. Compared with normal pregnancy, the expression of annexin A2 in placental trophoblast of preeclampsia cell surface is reduced.2 The expression level of annexin A2 in placental tissue and peripheral blood. Placental tissue and peripheral blood levels of annexin A2: early-onset mild preeclampsia group were 0.77±0.15 and 0.63±0.09, early-onset severe preeclampsia group were 0.76±0.19 and 0.66±0.13, late-onset mild preeclampsia group were 0.74±0.23 and 0.71±0.23, late-onset severe preeclampsia group were 0.81±0.35 and 0.59±0.14, and healthy pregnant group were 1.82±0.18 and 0.50±0.04. Compared with normal pregnancy, annexin A2 in preeclampsia is significantly reduced.3 The expression of annexin A2 mRNA by RT-PCR in placental tissue: early-onset mild preeclampsia group 27.94±1.49, early-onset severe preeclampsia group was 27.23±1.26, late-onset mild preeclampsia group was 28.27±0.85, late-onset severe preeclampsia group was 28.54±0.65, and healthy pregnancy group was 32.34±0.71.Conclusion: Annexin A2 mainly expresses on the surface of trophoblast cell. Compared with normal pregnancy, annexin A2 in placental of preeclampsia was decreased. It plays an important role in the occurrence and the development of preeclampsia. Part four The purification of annexin A5 and its anticoagulantion in vitroObjective: To investigate the anticoagulation of annexin A5 in vivo and in vitro.Methods: Placental tissues of healthy pregnancy women were collected for purification. The anticoagulation of annexin A5 was measured in vitro with different concentrations of annexin A5.Results:1 When the concentration of annexin A5≥30μg/ml, APTT gradually extended. There are significant differences between the groups.2 Compared with the normal group, the coagulation of peripheral blood was no significant difference.Conclusion: Annexin A5 anticoagulant activity is closely related with its concentration. When the concentration of annexin A5≥30μg/ml, APTT extended with the increasing concentration of annexin A5. However, the APTT in peripheral blood of patients with preeclampsia did not change. Part five Antibody detection in preeclampsiaObjective: To detect related antibodies in maternal blood of the preeclampsia and analyze the ralationship between of antibody and pathogenesis of preeclampsia.Methods: The level of anti-annexin A5, anti-annexin A2, anti-β2GPI and ACL in maternal blood of preeclampsia and normal pregnancy were dectected by ELISA.Results:1 Compared with normal pregnancy, levels of anti-annexin A5 in the peripheral blood of patients with preeclampsia is increased. But there was no significant difference between them (P=0.83).2 Compared with normal pregnancy, levels of anti-annexin A2 in the peripheral blood of patients with preeclampsia are increased, and it has a significant difference between them (P=0.04).3 Compared with normal pregnancy, levels of anti-β2GPI in the peripheral blood of patients with preeclampsia are increased, and it has a significant difference between them (P=0.01).4 Compared with normal pregnancy, ACL-positive rate in preeclampsia was increased.Conclusion:The antibody in peripheral blood, which binding to antigen antibody decreased the level of annexin A5, annexin A2 expression. And they influcene the coagulation and fibrinolysis system, which creats hypercoagulation of PE.更多还原