【作者】 徐京晶；

【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】 第一部分子痫前期胎盘滋养细胞中MMP-9及FasL表达的研究目的探讨MMP-9和FasL在子痫前期发病机制中的作用及两者间的关联。方法采用免疫组织化学法检测48例子痫前期孕妇(重度子痫前24例,轻度子痫前期24例)及24例正常晚期妊娠孕妇胎盘中MMP-9和FasL的表达定位及强度。结果MMP-9主要表达于胎盘合体滋养细胞细胞质和细胞膜内,FasL主要表达于胎盘合体滋养细胞细胞质和细胞膜内。轻、重度子痫前期组MMP-9表达水平明显低于正常晚孕组(P<0.05)。轻、重度子痫前期组FasL表达水平明显高于正常晚孕组(P<0.05)。胎盘组织中MMP-9与FasL表达水平呈负相关(r=-0.700,P<0.05)。结论1、子痫前期胎盘滋养细胞中MMP-9与FasL表达异常,使胎盘形成时滋养细胞入侵减少及血管重塑障碍,可能与子痫前期的发病机制有关。2、子痫前期胎盘滋养细胞中MMP-9与FasL的表达水平呈负相关,可能滋养细胞的侵入与其致内皮细胞凋亡作用之间存在关联,共同参与子痫前期的发生。第二部分绒毛外滋养细胞中MMP-9表达对FasL分泌的作用和机制的研究目的通过脂质体转染MMP-9siRNA,探讨绒毛外滋养细胞株TEV-1中基质金属蛋白酶-9(MMP-9)对可溶性Fas配体(sFasL)表达的调控。方法1、化学合成MMP-9siRNA,通过脂质体转染绒毛外滋养细胞株TEV-1。2、RealTime RT-PCR、ELISA检测MMP-9siRNA转染效率,ELISA检测细胞内、外FasL蛋白表达的变化。3、给予外源性MMP-9,ELISA检测细胞内、外FasL蛋白表达的变化。结果1、转染后,MMP-9 siRNA组的MMP-9 mRNA表达量与对照组相比显著降低,(P<0.05); MMP-9 siRNA组的FasL mRNA表达量与对照组相比差异无显著性意义(P>0.05)。2、转染后,MMP-9 siRNA组的MMP-9蛋白分泌表达与对照组中的表达量相比明显降低,(P<0.05); MMP-9 siRNA组细胞内的MMP-9蛋白表达与对照组中的表达量相比明显降低(P<0.05)。转染后,MMP-9 siRNA组的FasL蛋白分泌表达与对照组中的表达量相比明显降低,(P<0.05)；而MMP-9 siRNA组细胞内的FasL蛋白表达与对照组中的表达量相比明显增多,(P<0.05)。3、外源性MMP-9蛋白作用后,MMP-9 siRNA组及对照组中细胞外分泌的FasL蛋白表达均较作用前明显增多(P<0.05)；而MMP-9 siRNA组及对照组中细胞内的FasL蛋白表达均较作用前明显减少(P<0.05)。结论1、化学合成的MMP-9 siRNA能有效、特异性沉默TEV-1细胞株中的MMP-9基因。2、TEV-1细胞中MMP-9蛋白分泌表达影响细胞内、外FasL蛋白表达。3、外源性MMP-9蛋白也影响TEV-1细胞内、外FasL蛋白表达。第三部分MMP-9在绒毛外滋养细胞致内皮细胞凋亡中的作用和机制的研究目的通过脂质体介导转染MMP-9 siRNA,探讨绒毛外滋养细胞基质金属蛋白酶-9(MMP-9)的表达对血管内皮细胞凋亡的影响。方法1、在transwell上室培养TEV-1细胞株,并以脂质体介导转染MMP-9 siRNA及其对照至TEV-1。2、在transwell下室培养EVC-304细胞株,使TEV-1和EVC-304非接触性共培养。3、共培养后收集下室细胞及上清液,流式细胞仪检测EVC-304细胞的凋亡率。4、单独培养EVC-304,给予外源性MMP-9,流式细胞仪检测不同时间段EVC-304的凋亡率。结果1、共培养体系中,MMP-9 siRNA组的滋养细胞所致内皮细胞凋亡率比对照组有明显降低(P<0.05)；在MMP-9 siRNA+MMP-9组,内皮细胞凋亡率较MMP-9 siRNA组明显增多(P<0.05)；对于对照+MMP-9组,内皮细胞凋亡率较对照组明显增多(P<0.05)。2、MMP-9直接单独作用于内皮细胞后,不同时间段的内皮细胞凋亡率无显著性差异(P<0.05)。结论1、绒毛外滋养细胞分泌MMP-9的改变导致其诱导内皮细胞凋亡的改变。2、滋养细胞分泌MMP-9致内皮细胞凋亡率改变,是通过MMP-9对滋养细胞分泌FasL蛋白的调控实现的。更多还原

【Abstract】 Part one Study of expression of MMP-9 and FasL in placenta of preeclampsiaObjective:To investigate the involement of MMP-9 and FasL in the pathological mechanism of preeclampsia and the correlation between them.Methods:The expression of MMP-9 and FasL of placenta tissue in 24 cases of normal term preganancy and 48 cases of preeclampsia women was determined by immunohistological method.Results:Expression of MMP-9 and FasL was mainly located in the cytoplasm and membrane of trophoblasts. Compared with control group, the expression of MMP-9 in the preeclampsia group was significantly lower(P<0.05), while the expression of FasL in the preeclampsia group was significantly higher than those of control group(P< 0.05). There was a significantly negative correlation between the expression of MMP-9 and FasL in placenta tissue(r=-0.700,P<0.05).Conclusions:1. Altered expression of MMP-9 and FasL in trophoblasts might influence the invasion of trophoblasts and artery remodeling, thus contribute to the pathogenesis of preeclampsia.2. Trophoblast invasion might be associated with endothelial apoptosis, both of which were involved in the mechanism of preeclampsia. Part twoStudy of role and mechanism of MMP-9 of extravillous trophoblasts on FasL secretionObjective:To investigate the release of FasL by regulation of MMP-9 secreted by extravillous trophoblasts(TEV-1), MMP-9siRNA was transfected into TEV-1 by lipofectamine.Methods:1. MMP-9 siRNA was constructed and then transfected into TEV-1 cells by lipofectamine.2. After transfection, the level of MMP-9, FasL mRNA were assessed by real-time PCR, and the intracellular or extracellular level of these proteins were measured by ELISA.3. After administration of recombinant MMP-9, the intracellular or extracellular level of FasL was measured by ELISA.Results:1. The cells transfected with MMP-9 siRNA had a remarkable decrease in MMP-9 mRNA level as compared with those transfected with control siRNA(P<0.05), whereas FasL mRNA level between the two groups had no significant changes(P>0.05).2. MMP-9 siRNA caused a significant decrease in the secretion of MMP-9(P<0.05), whereas control siRNA did not alter the secretion. There was a decrease expression of intracellular MMP-9 in MMP-9 siRNA transfected cells when compared with cells transfected with control siRNA(P<0.05).The level of sFasL were significantly reduced in cells transfected with MMP-9 siRNA as compared with that in cells transfected with control siRNA(P<0.05). Increased expression of intracellular FasL was detected in cells transfected with MMP-9 siRNA as compared with that in cells with control siRNA(P<0.05).3. sFasL release was markedly recovered by adding exogenous MMP-9 to MMP-9 siRNA transfected cells(P<0.05). Following recombinant MMP-9 added to control siRNA, a significant increase of sFasL was present with exogenous MMP-9 treatment(P<0.05). Expression of intracellular FasL was recovered to control level after adding exogenous MMP-9 to MMP-9 siRNA treated cells(P<0.05). Exogenous MMP-9 to control siRNA was observed to cause significant decrease of intracellular FasL expression(P<0.05).Conclusions:1. MMP-9 siRNA efficiently and specifically degraded MMP-9 mRNA in TEV-1 cells.2. MMP-9 secreted by TEV-1 cells regulated both secreted and soluble FasL protein.3. Recombinant MMP-9 effected both secreted and soluble FasL protein in TEV-1 cells.Part threeStudy of role and mechanism of MMP-9 of extravillous trophoblasts on endothelial apoptosisObjective:To investigate the involvement of MMP-9 in trophoblasts-induced endothelial apoptosis, MMP-9siRNA was transfected into extravillous trophoblasts (TEV-1) by lipofectamine.Methods:MMP-9siRNA was transfected into extravillous trophoblasts (TEV-1) by lipofectamine. Then trophoblasts transfected with MMP-9siRNA or control siRNA were cocultured with endothelial cells respectively in a transwell system. The apoptosis of endothelial cells were detected by flow cytometry. Besides, EVC-304 cells were cultured under MMP-9 for different period of time to observe whether MMP-9 has direct apoptosis effect on EVC-304 cells.Results:1. The percentage of cells externalizing phosphatidylserine, quantified by flow cytometry, showing that the level of endothelial apoptosis were significantly decreased when cocultured with trophoblasts transfected with MMP-9 siRNA, as compared with that when cocultured with trophoblasts transfected with control siRNA(P<0.05). Yet, the apoptosis level was recovered to control level by adding MMP-9 back to MMP-9 silencing cells(P<0.05). It showed MMP-9 administration(5ng/mL) of TEV-1 cells caused increased endothelial apoptosis(P<0.05).2. We also studied whether MMP-9 alone, in addition to FasL, directly induces endothelial apoptosis. Apoptosis effect was investigated after stimulation of the cultures with MMP-9 (5ng/mL) for 6,12, or 24 hours. No difference was found as compared with control(P>0.05).Conclusions:1. MMP-9 secreted by extravillous trophoblasts induced endothelial apopotosis.2. MMP-9 alone has no direct apoptosis effect on endothelial cells, and MMP-9 secreted by trophoblasts contributes to endothelial apoptosis via extracellular release of soluble FasL.更多还原