【作者】 赵君；

【导师】 乔福元；

【作者基本信息】 华中科技大学 ， 妇产科学， 2011， 博士

【摘要】 第一部分重度子痫前期肾小球滤过屏障损伤的临床研究目的：探讨重度子痫前期肾小球滤过屏障损伤的相关情况及对妊娠结局的影响。方法：回顾性分析112例重度子痫前期患者的临床资料,了解重度子痫前期血清肌酐水平及尿蛋白丢失的程度。按尿蛋白丢失的程度分为随机尿蛋白(-)～(++)组(A组)30例,随机尿蛋白(+++)～(++++)组(B组)82例,对两组的血清肌酐水平、母体妊娠结局及围生儿结局进行比较。采用SPSS17.0软件进行统计学分析,计量资料采用t检验,计数资料采用卡方检验,当卡方检验中理论频数<1时,采用Fisher精确概率法检验。结果：在重度子痫前期患者中,血清肌酐升高者占总例数的30.4%,随机尿蛋白(+++)～(++++)者占总例数的73.2%,随着尿蛋白丢失的加重,血青肌酐含量显著升高(P<0.05),母体不良结局的总例数显著增加(P<0.05),胎儿窘迫的发生率显著升高(P<0.05)结论：重度子痫前期肾小球滤过屏障损伤及滤过功能减退均较常见,该损伤可能最终导致不良妊娠结局。第二部分滋养细胞通过产生sFlt-1引起肾小球内皮细胞屏障功能损伤的研究目的：1.研究滋养细胞大量表达sFlt-1对肾小球内皮细胞屏障功能的影响,探讨子痫前期VEGF与sFlt-1失衡对肾小球内皮细胞屏障功能的影响。2.研究VEGF对滋养细胞过表达sFlt-1引起的肾小球内皮细胞屏障功能损伤的影响,探讨VEGF在维护肾小球内皮细胞屏障功能中发挥的作用。方法：1.脂质体转染法将sFlt-1表达质粒转染到滋养细胞中,采用RT-PCR及real-time PCR方法监测滋养细胞中sFlt-1 mRNA的表达,并用ELISA方法检测滋养细胞中sFlt-1蛋白的表达,以确定转染效果。2.利用Transwell共培养模型将过量表达sFlt-1的滋养细胞与肾小球内皮细胞共培养,采用荧光标记的牛血清白蛋白检测肾小球内皮细胞的单层屏障功能。3.利用VEGF干预过量表达sFlt-1的滋养细胞和肾小球内皮细胞共培养组,采用荧光标记的牛血清白蛋白检测内皮细胞的单层屏障功能。结果：1.滋养细胞转染sFlt-1表达质粒后,滋养细胞中sFlt-1 mRNA及蛋白的表达显著升高。2.当过量表达sFlt-1的滋养细胞与肾小球内皮细胞共培养时,肾小球内皮细胞单层屏障功能明显受损。3.VEGF可显著改善过量表达sFlt-1的滋养细胞与肾小球内皮细胞共培养引起的肾小球内皮细胞单层屏障功能损伤。结论：滋养细胞中VEGF与sFlt-1失衡可引起肾小球内皮细胞屏障功能损伤,这可能是子痫前期肾小球内皮细胞屏障功能损伤的重要原因,VEGF在缓解由sFlt-1表达增加引起的肾小球内皮细胞屏障功能损伤方面起着至关重要的作用。第三部分凋亡／坏死滋养细胞导致肾小球内皮细胞屏障功能损伤的研究目的：1.研究凋亡、坏死滋养细胞对肾小球内皮细胞屏障功能的影响,探讨子痫前期肾小球内皮细胞屏障功能损伤的机制。2.研究VEGF对凋亡、坏死滋养细胞引起的肾小球内皮细胞屏障功能损伤的影响,探讨VEGF在肾小球内皮细胞屏障功能损伤机制中的作用。方法：1.利用紫外线照射诱导滋养细胞凋亡,利用水浴加温诱导滋养细胞坏死。采用流式细胞术及荧光染料染色等方法分析滋养细胞的凋亡与坏死。2.将正常、凋亡及坏死的滋养细胞和肾小球内皮细胞于Transwell系统上室中混合培养,采用荧光标记的牛血清白蛋白检测肾小球内皮细胞的单层屏障功能。3.利用VEGF干预凋亡、坏死的滋养细胞和肾小球内皮细胞混合培养组,采用荧光标记的牛血清白蛋白检测内皮细胞的单层屏障功能。4.分别用细胞示踪剂将滋养细胞与肾小球内皮细胞标记为不同颜色,将已标记的凋亡的滋养细胞与已标记的肾小球内皮细胞混合培养24小时,荧光显微镜下观察两种细胞的形态变化。结果：1.紫外线照射使滋养细胞的凋亡率达到80%以上,水浴加热使滋养细胞的坏死率达到80%以上。2.当正常、凋亡及坏死的滋养细胞与肾小球内皮细胞混合培养时,肾小球内皮细胞单层屏障功能明显受损。3.VEGF可显著改善凋亡、坏死的滋养细胞与肾小球内皮细胞混合培养引起的肾小球内皮细胞单层屏障功能损伤,但是不能使其屏障功能恢复到正常状态。4.肾小球内皮细胞可以吞噬凋亡的滋养细胞。结论：当滋养细胞直接作用于肾小球内皮细胞时,VEGF并不能完全改善肾小球内皮细胞的屏障功能损伤,这可能与肾小球内皮细胞吞噬滋养细胞有关。肾小球内皮细胞吞噬血液循环的滋养细胞可能是子痫前期肾小球屏障功能损伤的机制之一。更多还原

【Abstract】 Part I Clinical study on the barrier injury of glomerular filtration in severe preeclampsiaObjective:To investigate glomerular filtration barrier injury and its influence on pregnancy outcomes in severe preeclampsia.Methods:There were 112 cases of severe preeclampsia were analysed retrospectively. Increased serum creatinine and the severity of proteinuria were analyzed. All patients were divided into 2 groups according to their degree of proteinuria.30 patients who had urine dipstick protein values of 1+to 2+ were defined as Group A, and other 82 patients were in the group of 3+ to 4+ proteinuria on dipstick (Group B). The creatinine level, maternal pregnancy outcomes and perineal infant outcomes were compared in two groups. The data was analysised by SPSS 17.0 with t test, X2 test and Fisher’s exact probability test.Results:34 cases (30.4%) of the total cases were with increasing serum creatinine. Grade 3-4 proteinuria accounted for 73.2% of severe preeclampsia. The concentration of serum creatinine was elevated obviously, and the number of maternal pregnancy outcomes was significantly increased, and the incidence of fetal distress was higher obviously with the severity of proteinuria.Conclusion:The injury of glomerular filtration barrier and glomerular filtration dysfunction should be common and might induce adverse outcomes. PartⅡTrophoblasts derived sFlt-1 may contribute to the barrier dysfunction of glomerular endothelial cellsObjective:1. To investigate the effect of upregulated soluble fims-like tyrosine kinase receptor 1 (sFlt-1) in trophoblast cells on glomerular endothelial cells barrier function, in order to study the influence of the dysregulation of vascular endothelial growth factor (VEGF) and sFlt-1 on glomerular endothelial cells barrier function in preeclampsia.2. To investigate the effect of VEGF on the barrier dysfunction of glomerular endothelial cells by sFlt-loverexpression induced in trophoblasts, in order to study VEGF playing a role in maintaining glomerular endothelial cells barrier function.Methods:1. The human sFlt-1 expressing plasmid DNA was transfected into human first-trimester extravillous trophoblast cell line (TEV-1) by using Lipofectin method, and the levels of sFlt-1 mRNA and protein in TEV-1 were determined by performing RT-PCR, real-time PCR and ELISA.2. Transwell system was used to perform the coculture of TEV-1 s treated with transfection and human conditionally immortalised glomerular endothelial cell line (ciGEnCs). The monolayer barrier function of ciGEnCs was evaluated by transferring fluorescently-labeled BSA across ciGEnCs monolayer3. The cocultivation of TEV-1s treated with transfection and ciGEnCs was interfered with VEGF. The monolayer barrier function of ciGEnCs was evaluated by transferring fluorescently-labeled BSA across ciGEnCs monolayerResults:1. The expression of sFlt-1 at both mRNA and protein were enhanced after TEV-1cells were transfected with sFlt-1 plasmid DNA. 2. There was a significant increase of the amount of transferred fluorescently-labeled BSA across ciGEnCs monolayer in coculture of ciGEnCs with TEV-1 cells transfected with sFlt-1 plasmid DNA, and the monolayer barrier function of ciGEnCs was noticeably destroyed.3. Administration of VEGF could protect the monolayer barrier function of ciGEnCs as compared to that of cocultivation of TEV-1s transfected with sFlt-1 plasmid DNA and ciGEnCs.Conclusion:The dysregulation of VEGF and sFlt-1 in trophoblast may induce the barrier dysfunction of glomerular endothelial cells,which may be an important factor to contribute to glomerular endothelial cells barrier dysfunction in preeclampsia.VEGF may play an important role in relieving glomerular endothelial cells barrier dysfunction by sFlt-1 overexpression in trophoblast.PartⅢTrophoblasts induced apoptosis and necrosis may contribute to the barrier dysfunction of glomerular endothelial cellsObjective:1. To investigate the effect of both apoptotic and necrotic trophoblast cells on glomerular endothelial cells barrier function, in order to further study the mechanism of the barrier dysfunction of glomerular endothelial cells in preeclampsia.2. To investigate the effect of VEGF on the barrier dysfunction of glomerular endothelial cells which both apoptotic and necrotic trophoblasts caused, in order to study VEGF playing a role in maintaining glomerular endothelial cells barrier function. Methods:1. TEV-1 cells were induced to undergo apoptotic death by exposure to UV light and induced into necrosis by placing them into a 56℃water bath. Apoptotic and necrotic TEV-1 cells were analyzed by flow cytometry and Annexin V/PI double staining.2. CiGEnCs were inoculated in the upper compartment of the Transwell body until confluent was obtained. Subsequently, TEV-1 cells treated with control medium, apoptosis and necrosis were applied on confluent monolayers of ciGEnCs, respectively.The monolayer barrier function of ciGEnCs was evaluated by transferring fluorescently-labeled BSA across ciGEnCs monolayer3. The cocultivation of TEV-1s treated with apoptosis and necrosis and ciGEnCs was interfered with VEGF. The monolayer barrier function of ciGEnCs was evaluated by transferring fluorescently-labeled BSA across ciGEnCs monolayer4. TEV-1 cells treated with apoptosis were labeled with Dil cell-labeling solution, and ciGEnCs were labeled with Dil cell-labeling solution. Dil-labeled apoptotic or necrotic trophoblasts were incubated with the confluent monolayers of ciGEnCs for 24 h. Then, the cells were examined under fluorescence microscopeResults:1. More than 80% of the TEV-1 cells were apoptotic by exposure to UV light, and more than 80% of the TEV-1 cells were necrotic by water bath.2. When TEV-1s with medium, apoptosis and necrosis were cultured on confluent monolayers of ciGEnCs, the amounts of transferred fluorescently-labeled BSA across ciGEnCs monolayers were increased significantly.3. Excess of VEGF could protect the monolayer barrier function of ciGEnCs as compared to that of cocultivation of TEV-1s with apoptosis and necrosis and ciGEnCs, but could not restore ciGEnCs barrier function to its normal state.4. Apoptotic TEV-1 cells could be phagocytosed by ciGEnCs.Conclusion:When trophoblasts directly acted on glomerular endothelial cells, VEGF could not relieve completely the barrier dysfunction of glomerular endothelial cells, which may relate to the phagorytosis of ciGEnCs.Deported trophoblasts in the maternal blood phagocytosed by glomerular endothelial cells may be one of the mechanisms of glomerular endothelial cells barrier dysfunction in preeclampsia.更多还原