【作者】 芮广海；

【导师】 尚涛；

【作者基本信息】 中国医科大学 ， 妇产科学， 2007， 博士

【摘要】 内毒素对细胞滋养细胞浸润能力的调节作用及其与子痫前期的关系前言子痫前期是导致孕产妇和围产儿死亡和患病的主要原因之一,其发病机制至今仍不完全清楚。对子痫前期发病机制的研究是目前围产医学领域内的热点,已经取得了一些进展,并形成了多种学说。胎盘缺血学说是其中最为重要的学说之一。这种学说认为,妊娠期各种原因导致的滋养细胞浸润能力降低,使胎盘形成和植入障碍,引起子宫肌层内螺旋小动脉的重铸失败,胎盘血流灌注不足,不能满足胎儿生长发育的生理需求,出现胎盘缺血缺氧。随后造成胎盘组织释放大量毒性物质,出现全身性母体血管内皮细胞损伤和功能紊乱,最终母体产生蛋白尿、高血压、水肿等一系列临床症状,形成子痫前期。滋养细胞浸润能力受到多种因素的调节,目前认为,基质金属蛋白酶的表达减少和细胞过度凋亡是子痫前期细胞滋养细胞浸润能力下降的重要原因。基质金属蛋白酶是一组依赖Zn的蛋白水解酶家族,几乎水解所有的细胞外基质。其中MMP-2和MMP-9对细胞外基质的降解能力最强。在滋养细胞浸润过程中,一系列的母体细胞和它们的基底膜以及细胞外基质是滋养细胞必须穿过的结构。这些结构中的Ⅳ型胶原、纤维蛋白原、层粘连蛋白、弹性蛋白、巢蛋白和明胶及蛋白聚糖是MMP-2和MMP-9的底物。现已发现,与正常妊娠相比,子痫前期胎盘滋养层细胞MMP-2和MMP-9的表达明显减少。细胞凋亡也是影响细胞滋养细胞浸润能力的重要因素。在生理功能正常的情况下,形成和维持适度的凋亡是保持细胞滋养细胞正常浸润能力的基本前提。细胞过度凋亡将导致有浸润能力的细胞滋养细胞大量减少,导致胎盘形成和发育障碍。研究发现,子痫前期胎盘滋养细胞有过度凋亡和浸润不足发生,并认为这与子痫前期的发病机制有关。然而,目前并不清楚子痫前期细胞滋养细胞基质金属蛋白酶表达减少和过度凋亡发生的原因和机制。近年来的研究表明,内毒素与子痫前期的发生密切相关。内毒素存在于革兰氏阴性细菌细胞壁,在感染发生后,可被释放入机体。已发现妊娠期泌尿生殖系统、口腔感染是子痫前期重要的诱发因素,革兰氏阴性细菌是引起上述感染的主要菌种,而且,Fass等采用内毒素制备了大鼠的子痫前期模型。在此基础上,我们认为内毒素对细胞滋养细胞的浸润能力,即对基质金属蛋白酶的表达和细胞凋亡有调节作用。目的本文在体外实验和临床实验两个方面探讨内毒素对细胞滋养细胞浸润能力的影响;通过观察内毒素对细胞滋养细胞凋亡和基质金属蛋白酶的表达的调控,探讨内毒素对细胞滋养细胞浸润能力的调节作用;通过检测基质金属蛋白酶在子痫前期胎盘组织的表达,验证其与子痫前期的关系。方法从正常早期妊娠绒毛组织,分离细胞滋养细胞,并采用FD无血清培养基培养,然后采用RT-PCR和免疫荧光-激光共聚交检测内毒素对细胞滋养细胞表达基质金属蛋白酶的调节作用,并采用Transwell观察细胞滋养细胞在内毒素作用后浸润能力改变;采用电镜、Hoechst DNA染色、Western blot检测凋亡蛋白Bcl-2、Bax和caspase-8的表达等形态学和免疫学方法,观察内毒素对细胞滋养细胞凋亡的调节作用;最后,通过免疫组织化学方法研究基质金属蛋白酶在子痫前期与正常妊娠胎盘组织的表达。结果RT-PCR和免疫荧光-激光共聚交研究结果表明内毒素对基质金属蛋白酶在细胞滋养细胞的表达有明显抑制作用。经内毒素作用24h后MMP-2和MMP-9的mRNA和蛋白的表达明显减少。Transwell证实了内毒素对细胞滋养细胞浸润能力的抑制作用;细胞滋养细胞穿透Transwell基底膜的数量,对照组为143.1±18.4个;内毒素浓度为25、50、100、200ng/ml的各实验组分别为137.7±20.2个、122.8±17.5个、78.4±19.2个、50.1±23.5个,差异有统计学意义(P＜0.01)。形态学和免疫学研究表明内毒素对细胞滋养细胞有明确的凋亡诱导作用。在倒置显微镜下可见,滋养细胞呈单层生长,细胞呈多角形或长梭形,胞核呈圆形或椭圆形,胞浆透亮。经内毒素处理后,细胞形状变窄,胞浆透亮度降低,颗粒感增强,部分细胞突起回缩变圆,脱落呈悬浮状,并见细胞碎片。Hoechst染色DNA在荧光显微镜下观察对照组细胞核较大,染色质分布均匀;低浓度内毒素组见大部分均染、呈蓝色的细胞核,每视野可见1-3个染色质出现浓缩状态,偶见细胞核裂解;高浓度内毒素组多见染色质浓缩状态,细胞核裂解,可见凋亡小体。透射电镜观察可见凋亡细胞细胞质浓缩,细胞出芽,线粒体肿胀,细胞核的染色质高度凝聚、核膜裂解。Western blot结果表明内毒素抑制Bcl-2的表达,促进Bax和凋亡蛋白caspase-8在细胞滋养细胞的表达。内毒素作用24小时后各浓度组与对照组比较,滋养细胞Bcl-2、Bax和caspase-8的相对灰度值,差异有统计学意义(P＜0.01)。免疫组织化学研究结果表明,与正常妊娠相比,基质金属蛋白酶在子痫前期胎盘组织中的表达减少。结论本项研究表明,内毒素通过诱导细胞凋亡和抑制基质金属蛋白酶的表达来抑制细胞滋养细胞的浸润能力,进而阐明了妊娠期感染性因素与子痫前期发病机制之间的关系,同时也提示;在子痫前期的预防和治疗中,抗感染治疗有可能是一种重要手段,对其价值应进一步评估。更多还原

【Abstract】 Pre-eclampsia is the leading cause for mortality and morbidity of pregnancy and perinatal fetus. But the etiology and mechanism of pre-eclampsia is still uncertain. Therefore, the study on them is a focus in the field of perinatal medicine. Many advances have been taken up and many theories are developed. Hypoxia in placenta is one of the theories. The central point in it is that decreased invasion of cytotrophoblast results in abnormal development and placentation of placenta, and lead to failure of remodeling of the spiral artery and hypoxia in placenta. Hypoxia makes placenta tissue to release toxic substance, which can injury vascular endothelial cells and arise symptoms and signs of pre-eclampsia. Now it is suggested that decreased expression of matrix metalloproteinase and excessive apoptosis are leading cause for down-regulation of cytotrophoblast invasion.Matrix metalloproteinases are a family of Zn-dependent proteolytic enzymes, which hydrolyze almost all the extracellular matrix. Matrix metalloproteinases-2 and matrix metalloproteinases-9 are tightly connected with cytotrophoblast invasion. In the procession of invasion, cytotrophoblasts must break through the structure constituted of cells of the mother, its basement membrane and extracellular matrix. In thisstructure, there are plenty of type IV collagen, fibrinogen, laminin, vimentin,nidogen, gelatin and proteoglycans, which are substrates of matrix metalloproteinase-2 and matrix metalloproteinase-9.Recent studies indicated that endotoxin involves in pre-eclampsia. Endotoxin comes from the cell wall of gram-negative bacteria. When infection occurs, endotoxin enters human body. Now, urinogenital and oral infections are found to be predisposing factors of pre-eclampsia. In this infection, gram-negative bacteria are predominating. Moreover, Fass made a rat model of pre-eclampsia by injection endotoxin. According to the above discussion, we speculate that endotoxin has regulation effects on invasion of cytotrophoblast.ObjectiveThe aim of this study was to investigate effects of endotoxin on invasion of cytotrophoblast and its relationship with pre-eclampsia. In vitro experiments, we observed the effects of endotoxin on the expression of matrix metalloproteinase and apoptosis of cytotrophoblast. In clinical experiments, we revealed the relationship between the expression of matrix metalloproteinase in placenta and pre-eclampsia.MethodsThe expression of matrix metalloproteinase in cytotrophoblast treated with endotoxin was determined by RT-PCR and immune fluorescence-confocal microscope. Transwell was used to reveal the invasion of cytotrophoblast in vitro. In the observation of effects of endotoxin on apoptosis of cytotrophoblast, electron transmission microscope, Hoechst DNA staining and western blot were applied. Tissue immunochemistry was used to study the expression of matrix metalloproteinase in placenta in normal pregnancy and pre-eclampsia.ResultsThe result of RT-PCR and confocal microscope revealed that endotoxin inhibited expression of matrix metalloproteinase in cytotrophoblast. By using transwell, it was indicated that endotoxin inhibited invasion of cytotrophoblast. It was indicated that endotoxin induced apoptosis of cytotrophoblast in a dose-dependent manner by using morphological and immune methods. Immunochemistry study indicated that expression of matrix metalloproteinase in placenta was markly decreased in pre-eclampsia, in contrast to normal pregnancy.ConclusionsIn this study, endotoxin was found to down-regulate invasion of cytotrophoblast. It revealed the mechanism by which infection in pregnancy induced pre-eclampsia and the value of antibiotic in the prevention and treatment of pre-eclampsia.更多还原