GnT-V调控滋养细胞和血管内皮细胞功能在子痫前期发病中作用机制研究

发布时间：2018-07-28 18:24

【摘要】：目的:N-乙酰氨基葡萄糖转移酶V(N-acetylglucosaminyltransferase V,Gn T-V)是N-乙酰氨基葡萄糖转移酶家族中的一员,它主要作用是催化N-联接聚糖形成,调控细胞的生物学功能,参与细胞的粘附、迁移、侵袭和细胞内信号传导等过程,并发挥着重要的作用。以往关于Gn T-V的研究主要集中在与肿瘤的恶性程度的关系上;而有关于Gn T-V在人类胎盘中的研究很少。所以,本实验将从以下几个方面对Gn T-V在胎盘上的作用进行探讨:(1)首先,利用组织标本(正常早孕和晚孕胎盘,后者又包括Normal control组和PE组),检测Gn T-V定位的情况,和在PE中Gn T-V的表达是否有变化;(2)采用来自人绒毛外滋养细胞的细胞株HTR8/Svneo以及,人脐静脉内皮细胞株HUVECs(human umbilical vein endothelial cells)来检测Gn T-V的表达,研究Gn T-V对二者细胞生物学功能的调控;(3)对HTR8/Svneo细胞以及HUVECs进行H/R的处理,模拟PE初期的氧化应激损伤,探究Gn T-V基因是如何通过FAK-ERK信号通路调控滋养细胞以及内皮细胞的生物学功能,并最终引起PE发病的相关机制。方法:(1)通过IHC的SP法检测早孕期以及晚孕期胎盘组织中Gn T-V的表达水平、定位的情况。(2)采用WB和q RT-PCR研究Normal组和PE组Gn T-V的表达的变化。(3)通过慢病毒sh RNA转染,下调人滋养细胞株(HTR8/Svneo细胞)、脐静脉内皮细胞株(HUVECs)和早孕期绒毛外植体中Gn T-V的表达,并采用q RT-PCR、WB检测Gn T-V sh RNA的干扰效率,并使用FCM检测细胞的凋亡,MTT法检测细胞增殖情况。(4)通过Transwell法的迁移和侵袭实验下调了Gn-V后HTR8/SVneo、HUVECs细胞生物学功能的改变。(5)采用管腔成型实验检测Gn T-V是否影响HUVEC形成管样结构的潜能。(6)利用原代模型(绒毛外植体),评估Gn T-V对绒毛外滋养细胞的外生性迁移影响。(7)收集绒毛外植体和两种细胞的培养上清液,使用明胶酶谱法检测其中的MMP2/9活性;WB检测TIMP1/2的蛋白表达水平。(8)对HTR8/SVneo、HUVECs进行缺氧/复氧(H/R)处理,了解其对细胞生物学功能以及Gn T-V的表达情况的影响。(9)最后将细胞置于缺氧/复氧,的情况下,通过干扰Gn T-V基因及阻断其下游FAK-ERK信号通路观察细胞功能是否发生改变,从而了解Gn T-V调控细胞的功能在胎盘的发生和在PE发病中的作用和影响。结果:(1)通过IHC,我们发现Gn T-V在正常胎盘组织(早孕和晚孕)及PE组织中均有表达,主要定位于CTBs、STBs、TC中和血管内皮细胞(ECs)的胞浆内。而Gn T-V在蜕膜组织中,主要表达在EVTs和蜕膜细胞(DCs)中。且Gn T-V在PE组的表达较Normal组有升高的趋势。(2)在HTR8/Svneo细胞和HUVECs中干扰Gn T-V的表达,可促进前者的迁移和侵袭能力和后者的迁移和管样结构成型的潜能。(3)通过对HTR8/Svneo细胞以及HUVECs缺氧/复氧处理,可导致其功能减弱,伴随着Gn T-V表达上升。而我们还发现H/R处理使得了两个细胞中的FAK-ERK通路发生了明显活化。(5)使用sh RNA干扰Gn T-V基因表达和ERK1/2特异性的抑制剂PD98059阻断FAK-ERK通路,都对H/R所致的细胞功能受损有明显的改善。结论:(1)Gn T-V参与对滋养细胞和内皮细胞的细胞功能的调控,可能在整个妊娠期间都发挥着作用。(2)下调Gn T-V的表达促进HTR8/Svneo细胞的侵袭和迁移能力,促进HUVECs迁移及管样结构成型的潜能。(3)在氧化应激的损伤下,Gn T-V的表达上升,可通过调控FAK-ERK信号通路减弱滋养细胞的侵袭力和内皮细胞的管样结构成型的潜能,从而参与PE的发病。
[Abstract]:Objective: N- acetylglucosamine transferase V (N-acetylglucosaminyltransferase V, Gn T-V) is a member of the N- acetylglucosamine transferase family. Its main role is to catalyze the formation of N- join glycan, regulate the biological functions of cells, participate in cell adhesion, migration, invasion and intracellular signal transduction, and play an important role. The previous research on Gn T-V is mainly focused on the relationship with the malignancy of the tumor; and there are few studies on the Gn T-V in the human placenta. Therefore, this experiment will discuss the role of Gn T-V in the placenta from the following aspects: (1) first, using the group specimens (normal pregnancy and late pregnancy placenta, and the latter including Nor) The mal control group and the PE group) detected the location of Gn T-V and the change in the expression of Gn T-V in PE; (2) the expression of the cell line from human villous trophoblastic cells and the human umbilical vein endothelial cell line HUVECs (human umbilical) were used to detect the expression of Gn T-V. Regulation of energy; (3) H/R treatment of HTR8/Svneo cells and HUVECs to simulate oxidative stress damage at the early stage of PE, and to explore how the Gn T-V gene regulates the biological functions of trophoblastic and endothelial cells through FAK-ERK signaling and ultimately causes the pathogenesis of PE. Methods: (1) detection of early pregnancy and late pregnancy by IHC SP method. The expression level of Gn T-V in placenta tissue during pregnancy and localization. (2) the expression of Gn T-V in Normal and PE groups was studied by WB and Q RT-PCR. (3) the expression of human trophoblast (HTR8/Svneo cells), umbilical vein endothelial cell line and early pregnancy villi explants were downregulated by lentivirus sh RNA transfection. The interference efficiency of Gn T-V sh RNA was detected by WB, and cell apoptosis was detected by FCM, and cell proliferation was detected by MTT method. (4) the biological function of HUVECs cells was reduced by Transwell method migration and invasion experiments. (5) the potentiality of the formation of tubular like structure was detected by the lumen molding test. (6) Using the original model (villous explant) to evaluate the effect of Gn T-V on the exogenic migration of villous trophoblast cells. (7) collect the culture supernatant of villous explants and two cells, use gelatinase spectroscopy to detect MMP2/9 activity, and WB to detect the protein expression level of TIMP1/2. (8) HTR8/SVneo and HUVECs are treated with hypoxia / reoxygenation (H/R). The effect on the cell biological function and the expression of Gn T-V. (9) at last the cells were placed in anoxia / reoxygenation, and the changes in cell function were observed by interfering with the Gn T-V gene and blocking the downstream FAK-ERK signaling pathway, so as to understand the role of Gn T-V in regulating the function of fine cell in the occurrence of placenta and the role of Gn in the pathogenesis of PE. Results: (1) through IHC, we found that Gn T-V was expressed in normal placental tissue (early pregnancy and late pregnancy) and PE tissues, mainly located in the cytoplasm of CTBs, STBs, TC and vascular endothelial cells (ECs), and Gn T-V was mainly expressed in EVTs and decidual cells (DCs) in decidua. High trends. (2) the interference of Gn T-V expression in HTR8/Svneo cells and HUVECs can promote the migration and invasion of the former and the potential for the migration of the latter and the formation of the tube like structure. (3) through the treatment of HTR8/Svneo cells and HUVECs anoxia / reoxygenation, the function is weakened and the expression of Gn T-V increases. And we also find H/R treatment. The FAK-ERK pathway in the two cells was obviously activated. (5) the use of SH RNA interference with Gn T-V gene expression and ERK1/2 specific inhibitor PD98059 to block the FAK-ERK pathway, all of which have significantly improved the impairment of cell function caused by H/R. Conclusion: (1) Gn T-V participates in the regulation of cell function of nourishing and endothelial cells. (2) downregulation of Gn T-V expression promotes the invasion and migration of HTR8/Svneo cells and promotes the potential of HUVECs migration and tube like structure formation. (3) the expression of Gn T-V is increased under oxidative stress, and the invasiveness of trophoblastic cells and endothelial cells can be weakened by regulating FAK-ERK signaling pathway. The potential of structural formation to participate in the pathogenesis of PE.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R714.244

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