过度自噬与早发型子痫前期胎盘功能紊乱的关系

发布时间：2018-03-01 23:33

本文关键词： 过度自噬早发型子痫前期滋养细胞血管内皮细胞胎盘功能紊乱　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：本文通过检测人胎盘滋养细胞和血管内皮细胞中自噬相关蛋白微管相关蛋白1轻链3（microtubule-associated protein1light chain3，LC3）和Beclin-1的表达情况，证实早发型子痫前期胎盘中滋养细胞和血管内皮细胞发生过度自噬。进而，通过葡萄糖氧化酶（glucoseoxidase, GO）模拟体外氧化应激环境，以滋养细胞和血管内皮细胞为研究对象，探讨过度自噬参与早发型子痫前期（early-onsetpreeclampsia，EOPE）胎盘功能紊乱的病理及分子机制。方法：1.收集2010年1月至2012年5月在重庆医科大学附属第一医院产科收治的18例早发型子痫前期（34孕周）患者的胎盘组织和同期住院分娩的20例正常产妇的胎盘组织。应用免疫组化检测各组胎盘组织中滋养细胞和血管内皮细胞LC3和Beclin-1的表达；透射电镜观察两组胎盘组织中滋养细胞和血管内皮细胞自噬体的发生情况；组织免疫荧光共标法检测硝基酪氨酸（nitrotyrosine）和LC3、Beclin-1在胎盘组织中的表达关系。2.以GO模拟氧化应激环境，通过细胞免疫荧光法、实时荧光定量PCR（quantitative real-time polymerase chain reaction,qRT-PCR）及蛋白印记法（western blot, WB）方法测定经处理后的人绒毛外滋养细胞株（HTR8/SVneo）和人脐静脉内皮细胞株（humanumbilical vein endothelial cells, HUVECs）自噬发生的情况，透射电镜观察各组细胞中自噬体。然后通过hoechst33258观察各组细胞凋亡发生情况，WB检测各组细胞中cleaved caspase-3的表达情况。最后通过WB检测滋养细胞中基质金属蛋酶9（matrix metalloproteinase9,MMP9）和血管内皮细胞中血管内皮生长因子受体2（vascularendothelial growth factors receptor2, VEGFR2）表达情况，transwell侵袭实验检测滋养细胞细胞侵袭能力，管腔成形实验检测HUVECs的成管能力。结果：1. EOPE胎盘中自噬发生增加。（1）免疫组化提示早发型子痫前期胎盘滋养细胞和血管内皮细胞中LC3和Beclin-1表达量较正常胎盘增高（P 0.05）。（2）透射电镜在早发型子痫前期胎盘滋养细胞和血管内皮细胞中均找到典型的自噬体。（3）组织免疫荧光共标法发现EOPE胎盘中nitrotyrosine表达较正常胎盘增加，同时EOPE胎盘中LC3和Beclin-1表达增强。2.氧化应激诱导的过度自噬削弱HTR8/SVneo和HUVECs细胞功能。（1）细胞免疫荧光、qRT-PCR、WB均提示GO模拟氧化应激环境下HTR8/SVneo和HUVECs中GO组LC3和Beclin-1表达较正常组增加，差异有统计学意义（P 0.05）；同时通过透射电镜在HTR8/SVneo和HUVECs GO组中找到典型自噬体。（2）Hoechst33258染色提示各组细胞中凋亡的发生无明显差异，同时WB提示各组细胞中cleaved caspase-3表达无明显差异（P0.05）。（3）WB及细胞免疫荧光结果示HTR8/SVneo中GO组MMP9表达较其他组降低（P 0.05）；transwell侵袭实验结果示GO组HTR8/SVneo细胞侵袭力较其他组明显降低（P 0.05），3-MA+GO组细胞侵袭力较GO组增加（P 0.05）。HUVECs中，WB和细胞免疫荧光结果示GO组VEGFR2的表达较其他组降低（P 0.05），，3-MA+GO组VEGFR2表达较GO组增加（P 0.05）。管腔成形验结果示GO组HUVEC细胞成管能力较其他组降低（P 0.05)，3-MA+GO组细胞成管能力较GO组增加（P 0.05）。结论：1.早发型子痫前期胎盘中发生过度自噬。2.过度自噬降低HTR8/SVneo侵袭力和HUVECs管腔成形能力。3.滋养细胞和血管内皮细胞过度自噬可能是早发型子痫前期胎盘功能紊乱的另一重要发病机制。
[Abstract]:Objective: through the detection of autophagy of human placental trophoblast cells and vascular endothelial cells in protein microtubule associated protein 1 light chain 3 (microtubule-associated protein1light, chain3, LC3 and Beclin-1) expression, confirmed early onset preeclampsia placental trophoblast cells and vascular endothelial cells to excessive autophagy. Furthermore, the glucose oxidase (glucoseoxidase, GO simulation in vitro oxidative stress environment), to nourish cells and vascular endothelial cells as the research object, investigate the excessive autophagy in early onset preeclampsia (early-onsetpreeclampsia, EOPE) pathology and molecular mechanism of placental dysfunction.
Methods: 1. from January 2010 to May 2012 in First Affiliated Hospital of Chongqing Medical University from 18 cases of early-onset preeclampsia (34 weeks) placenta tissue and 20 cases of normal pregnancy placenta of patients and the same period of delivery in hospital. The expression of immunohistochemistry was detected in the placental tissue of trophoblast cells and vascular endothelial cells LC3 and Beclin-1 were observed; two groups of placenta trophoblast and endothelial cells autophagy of TEM; immunofluorescence is standard method for the detection of nitrotyrosine (nitrotyrosine) and LC3,.2. was simulated by GO under oxidative stress and the expression of Beclin-1 in placenta tissues, cells by immunofluorescence, real-time fluorescence quantitative PCR (quantitative real-time polymerase chain reaction, qRT-PCR) and Western blot (Western blot WB) method for the determination of the treated human extravillous trophoblast fine Cell line (HTR8/SVneo) and human umbilical vein endothelial cell line (humanumbilical vein endothelial cells, HUVECs) of autophagy, autophagy in the cells were observed by transmission electron microscope. Then through the body to observe the occurrence of apoptosis of hoechst33258 cells, the expression of WB was detected by cleaved caspase-3. Finally by trophoblast cells detection in WB metal matrix 9 eggs (matrix Metalloproteinase9, MMP9 enzyme) in vascular endothelial cells and vascular endothelial growth factor receptor 2 (vascularendothelial growth factors receptor2, VEGFR2) expression of Transwell and invasion capability of cell assay nourish, lumen into the tube forming experiment of HUVECs detection ability.
Results: 1. EOPE increased autophagy in the placenta. (1) immunohistochemistry showed early onset preeclampsia placental trophoblast cells and vascular endothelial cells LC3 and Beclin-1 expression of zfyve26in normal placenta (P 0.05). (2) transmission electron microscope were found in typical autophagosomes in early onset preeclampsia placental trophoblast cells and blood vessels endothelial cells. (3) immunofluorescence were found to increase the standard method than the normal placental nitrotyrosine expression of EOPE in placenta, EOPE and LC3 in placenta and the increased expression of Beclin-1 excessive autophagy in.2. induced oxidative stress and weaken the HTR8/SVneo HUVECs cell function. (1) immunofluorescence, qRT-PCR, WB showed that compared with normal GO simulation group LC3 and group GO increased the expression of Beclin-1 under oxidative stress of HTR8/SVneo and HUVECs, the difference was statistically significant (P 0.05); at the same time through transmission electron microscopy to find the typical autophagosomes in HTR8/SVneo and HUVECs (2 in the GO group. Hoechst33258 staining) prompt cell apoptosis was no significant difference in WB at the same time, suggesting that these cells cleaved caspase-3 expression had no significant difference (P0.05). (3) WB and cell immunofluorescence showed that HTR8/SVneo GO expression of MMP9 was lower than other groups (P 0.05); Transwell invasion experiment results show the GO group of HTR8/SVneo cells the invasion was significantly lower than the other groups (P 0.05), 3-MA+GO group of cell invasion were higher than that in GO group (P 0.05).HUVECs, WB expression and cell immunofluorescence showed that GO group VEGFR2 lower than other groups (P 0.05), 3-MA+GO group increased VEGFR2 expression compared with GO group (P 0.05). Lumen forming the results showed HUVEC cells GO group forming lower than other groups (P 0.05), 3-MA+GO group cell tube formation were higher than that in GO group (P 0.05).
Conclusion: excessive autophagy decreased HTR8/SVneo invasion and HUVECs lumen forming ability of.3. in trophoblast cells and vascular endothelial cells may be excessive autophagy in early onset preeclampsia placenta dysfunction is another important pathogenesis of excessive autophagy in.2. occurred in 1. early onset preeclampsia.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.255

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