【作者】 周琼；

【导师】 乔福元；

【作者基本信息】 华中科技大学 ， 妇产科学， 2010， 博士

【摘要】 目的：研究正常及子痫前期患者血清中可溶性血管内皮生长因子受体1(soluble fims-like tyrosine kinase receptor 1, sFlt-1)及血管内皮生长因子(vascular endothelial growth factor, VEGF)蛋白的表达差异,以及血清对内皮细胞损伤的影响,探讨子痫前期血清中sFlt-1及VEGF表达变化与内皮细胞损伤的关系。方法：1.采用酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)定量检测10例正常妊娠及10例重度子痫前期患者血清中sFlt-1及VEGF的蛋白表达水平。2.分别用上述10例正常妊娠及10例重度子痫前期患者血清在体外干预人脐静脉内皮细胞株(human umbilical vein endothelial cell lines, HUVEC)生长,利用：①荧光标记的牛血清白蛋白检测内皮细胞的单层屏障功能；②噻唑兰比色法(Methl thiazolyl tetrazolium,MTT)检测内皮细胞的增殖能力；③硝酸还原酶法检测内皮细胞的一氧化氮(nitric oxide, NO)合成能力；以评估两组血清对内皮细胞功能的影响。结果：1.重度子痫前期组患者血清中sFlt-1蛋白水平较正常孕妇组明显升高；而游离VEGF蛋白水平较正常孕妇组明显降低。重度子痫前期组血清中sFlt-1与游离VEGF蛋白水平呈负相关。2.与正常妊娠组相比,重度子痫前期组血清可使脐静脉内皮细胞功能明显受损,包括：①通过单层内皮细胞的牛血清白蛋白浓度升高,即内皮细胞的单层屏障功能受损；②内皮细胞增殖能力明显被抑止：③培养基中NO浓度明显降低。3.脐静脉内皮细胞功能损伤程度与重度子痫前期组血清中sFlt-1/VEGF浓度成正相关。结论：子痫前期患者血清中sFlt-1和VEGF表达失衡,与子痫前期内皮细胞损伤密切相关,可能是参与子痫前期发生发展的重要机制之一。目的：研究VEGF表达下调对内皮细胞损伤的影响,从细胞水平探讨VEGF在内皮细胞损伤机制中的作用。方法：1.利用siRNA (small interfering RNAs)干扰技术阻断人脐静脉内皮细胞中VEGF表达,采用逆转录聚合酶链反应(reverse transcription polymerase chain reaction, RT-PCR)和实时定量多聚酶链反应(real-time polymerase chain reaction, real-time PCR)检测VEGF基因表达,ELISA法检测培养基中VEGF蛋白表达。2.将VEGF-siRNA转染到人脐静脉内皮细胞中,利用：①荧光标记的牛血清白蛋白检测内皮细胞的单层屏障功能；②MTT法检测内皮细胞的增殖能力；③硝酸还原酶法检测内皮细胞的NO合成能力；以评估内皮细胞功能的变化。结果：1.VEGF-siRNA转染到人脐静脉内皮细胞48小时后,与control-siRNA组相比,VEGF mRNA表达明显降低(约50.7%)；同时,培养基中游离VEGF蛋白水平也明显降低。2.转染了VEGF-siRNA的脐静脉内皮细胞功能明显受损,包括：①通过单层内皮细胞的牛血清白蛋白浓度明显升高,即内皮细胞的单层屏障功能明显受损：②内皮细胞增殖能力显著被抑止；③培养基中NO浓度明显降低。这种内皮细胞功能改变与前述子痫前期组血清引起的内皮细胞功能改变相一致。结论：VEGF在维持正常的内皮细胞功能方面至关重要,VEGF表达下降可导致内皮细胞功能明显受损,这可能是引起子痫前期一系列临床症状的重要原因。目的：1.研究缺氧对滋养细胞及内皮细胞中sFlt-1表达的影响,探讨子痫前期sFlt-1表达异常的原因。2.研究缺氧滋养细胞对内皮细胞损伤的影响及其sFlt-1/VEGF损伤途径,从细胞水平深入探讨子痫前期滋养—内皮损伤机制。方法：1.利用二氯化钻分别诱导人早孕绒毛滋养细胞(human first-trimester extravillous trophoblast cell line, TEV-1)及脐静脉内皮细胞(human umbilical vein endothelial cell line, HUVEC)化学缺氧,模拟体外缺氧环境。采用RT-PCR及real-time PCR检测细胞中sFlt-1基因的表达,并用ELISA试剂盒检测培养基中sFlt-1蛋白的表达。2.利用二氯化钴诱导滋养细胞化学缺氧,并将缺氧的滋养细胞和内皮细胞于transwell系统中共培养。采用：①荧光标记的牛血清白蛋白检测内皮细胞的单层屏障功能；②MTT法检测内皮细胞的增殖能力；③硝酸还原酶法检测内皮细胞的NO合成能力；以评估内皮细胞功能的变化。同时,采用RT-PCR及real-time PCR检测滋养细胞中sFlt-1和VEGF基因的表达,并用ELISA试剂盒检测共培养的培养基中sFlt-1和VEGF蛋白的表达。3.利用VEGF干预缺氧滋养细胞和内皮细胞共培养组。采用：①荧光标记的牛血清白蛋白检测内皮细胞的单层屏障功能；②MTT法检测内皮细胞的增殖能力；③硝酸还原酶法检测内皮细胞的NO合成能力；以评估内皮细胞功能的变化。结果：1.缺氧处理0,24,48,72,96,120小时,滋养细胞中sFlt-1基因及蛋白表达随缺氧时间的延长明显增加(mRNA:72,96,120小时P<0.05；蛋白96,120小时P<0.05)；而内皮细胞中sFlt-1基因及蛋白表达随缺氧时间的延长则无明显改变。2.当缺氧的滋养细胞与内皮细胞共培养时,内皮细胞功能明显受损,包括：①内皮细胞的单层屏障功能损伤；②内皮细胞增殖能力显著被抑止；③培养基中NO浓度明显降低。同时,共培养的缺氧滋养细胞中sFlt-1及VEGF基因表达明显升高；共培养的培养基中sFlt-1蛋白表达升高,但VEGF蛋白表达却明显降低。3.加入VEGF可显著改善上述共培养引起的内皮细胞损伤,包括内皮细胞单层屏障功能、细胞增殖能力及NO合成能力均明显提高。结论：慢性缺氧的滋养细胞可高表达sFlt-1,可能是子痫前期sFlt-1升高的重要来源。sFlt-1可作为“胎盘来源的毒性因子”,通过降低VEGF活性,从而参与了子痫前期内皮细胞的损伤,更多还原

【Abstract】 Objective:To investigate the expression of soluble fims-like tyrosine kinase receptor 1 (sFlt-1) and vascular endothelial growth factor (VEGF) in serum obtained from normal pregnancy and preeclampsia, and the correlation to endothelial cell dysfunction.Methods:1. The level of sFlt-1 and VEGF protein in serum samples of 10 severe preeclampsia females and 10 normotensive females were determined by performing enzyme-linked immunosorbent assay (ELISA).2. The culture of human umbilical vein endothelial cell lines (HUVEC) was interfered with serum samples as described previously. The effect of serum on endothelial cell dysfunction was determined on the basis of following aspects:①monolayer barrier function was evaluated by transferring fluorescently-labeled BSA across HUVEC monolayer;②cell proliferation function was evaluated by performing methl thiazolyl tetrazolium (MTT);③level of secreted nitric oxide (NO) was estimated by nitrate reductase assay.Result:1. The level of sFlt-1 protein in serum of severe preeclampsia was significantly higher than that of normal pregnancy, whereas the level of VEGF protein was significantly lower. There was obvious negative correlation between the level of sFlt-1 and VEGF in preeclampsia.. 2. Compared to normal pregnancy, the serum of severe preeclampsia could lead to endothelial cell dysfunction, including:①high transfer of fluorescently-labeled BSA across HUVEC monolayer indicated the damage of monolayer barrier function;②the proliferation ability of HUVEC was markedly decreased;③the level of secreted NO was low as compared to that of normal pregnancy group.3. There was positive correlation between endothelial cell dysfunction and the level of sFlt-1/VEGF in serum of preeclampsia.Conclusion:The results of our study suggest that endothelial cell dysfunction is closely correlated with disorder of sFlt-1 and VEGF levels in serum of preeclampsia, which may lead to the pathogenesis of preeclampsia.Objective:To investigate the effects of VEGF deficit on endothelial cell dysfunction, in order to study the role of VEGF in pathogenic mechanism of endothelial dysfunction in preeclampsia.Methods:1. VEGF expression was blocked by transfecting HUVEC with VEGF-siRNA. The level of VEGF protein and mRNA in transfected HUVEC was determined by performing reverse transcriptase-polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunosorbent assay (ELISA).2. The effect of VEGF deficit on endothelial cell dysfunction was determined on the basis of following aspects:①monolayer barrier function was evaluated by transferring fluorescently-labeled BSA across HUVEC monolayer;②cell proliferation function was evaluated by performing MTT;③level of secreted NO was estimated by nitrate reductase assay.Result:1. After transfecting HUVEC with VEGF-siRNA for 48 h, VEGF mRNA expression was significantly reduced as compared to the expression in scramble siRNA-transfected cells (approximately 50.7%). In addition, the concentration of VEGF protein secreted by VEGF-siRNA transfected cells was lower than that of scramble-siRNA treated cells.2. The function of VEGF-siRNA transfected HUVEC was destroyed, including:①significantly high transfer of fluorescently-labeled BSA across HUVEC monolayer indicated the badly damage of monolayer barrier function;②the proliferation ability of HUVEC was markedly decreased;③the level of secreted NO was low as compared to that of scramble siRNA-transfected cells.Conclusion:VEGF plays an important role in maintaining the physiological functions of endothelial cells. The deficiency of VEGF can lead to endothelial cell dysfunction, which may induce a series of clinical symptom in preeclampsia.Objective:1. To investigate the effects of hypoxia on sFlt-1 expression respectively in trophoblast cell and endothelial cell, in order to study the reason for abnormal expression of sFlt-1 in preeclampsia.2. To investigate the effect of hypoxia trophoblast-derived sFlt-1 on endothelial cell dysfunction by depriving of VEGF activity, in order to further study the mechanism of trophoblast-endothelial cell dysfunction in preeclampsia.Methods:1. Human first-trimester extravillous trophoblast cell line (TEV-1) and human umbilical vein endothelial cell line (HUVEC) were grown respectively in a hypoxic condition produced by COCl2. The levels of sFlt-1 mRNA and protein in TEV-1 and HUVEC were determined by performing RT-PCR, real-time PCR and ELISA.2. Transwell system was used to perform the cocultivation of hypoxia TEV-1 and HUVEC. The functions of HUVEC were evaluated on the basis of following aspects:①monolayer barrier function was evaluated by transferring fluorescently-labeled BSA across HUVEC monolayer;②cell proliferation function was evaluated by performing MTT;③level of secreted NO was estimated by nitrate reductase assay. Meanwhile, the levels of sFlt-1 and VEGF mRNA and protein in hypoxia TEV-1 were determined by performing RT-PCR, real-time PCR and ELISA.3. The cocultivation of hypoxia TEV-1 and HUVEC was interfere with VEGF, and the functions of HUVEC were evaluated on the basis of following aspects:①monolayer barrier function was evaluated by transferring fluorescently-labeled BSA across HUVEC monolayer;②cell proliferation function was evaluated by performing MTT;③level of secreted NO was estimated by nitrate reductase assay.Result:1. COCl2 showed time-dependent promotion on sFlt-1 mRNA (for 72,96 and 120 h treatment, P<0.05) and protein expression (for 96 and 120 h treatment, P<0.05) in TEV-1. However, there were not significant differences in sFlt-1 expression for HUVEC.2. When the cocultivation of hypoxia TEV-1 and HUVEC was performed, the functions of HUVEC was noticeably destroyed, including:①significantly high transfer of fluorescently-labeled BSA across HUVEC monolayer indicated the badly damage of monolayer barrier function;②the proliferation ability of HUVEC was markedly decreased;③the level of secreted NO was low as compared to that of normal cocultivation of TEV-1 and HUVEC. 3. Administration of VEGF could protect endothelium cell function from injury, which was determined on the basis of rising of monolayer barrier function, cell proliferation function, and secreted NO levels as compared to that of cocultivation of hypoxia TEV-1 and HUVEC.Conclusion:Chronic hypoxia trophoblast-derived sFlt-1 may be the important source of high expression of sFlt-1 in preeclampsia. Served as toxic factors derived from placenta, sFlt-1 is the key factors to contribute to endothelial cell dysfunction in preeclampsia by depriving of VEGF activity.更多还原