【摘要】：目的:N-乙酰氨基葡萄糖轉移酶V(N-acetylglucosaminyltransferase V,Gn T-V)是N-乙酰氨基葡萄糖轉移酶家族中的一員,它主要作用是催化N-聯接聚糖形成,調控細胞的生物學功能,參與細胞的粘附、遷移、侵襲和細胞內信號傳導等過程,并發(fā)揮著重要的作用。以往關于Gn T-V的研究主要集中在與腫瘤的惡性程度的關系上;而有關于Gn T-V在人類胎盤中的研究很少。所以,本實驗將從以下幾個方面對Gn T-V在胎盤上的作用進行探討:(1)首先,利用組織標本(正常早孕和晚孕胎盤,后者又包括Normal control組和PE組),檢測Gn T-V定位的情況,和在PE中Gn T-V的表達是否有變化;(2)采用來自人絨毛外滋養(yǎng)細胞的細胞株HTR8/Svneo以及,人臍靜脈內皮細胞株HUVECs(human umbilical vein endothelial cells)來檢測Gn T-V的表達,研究Gn T-V對二者細胞生物學功能的調控;(3)對HTR8/Svneo細胞以及HUVECs進行H/R的處理,模擬PE初期的氧化應激損傷,探究Gn T-V基因是如何通過FAK-ERK信號通路調控滋養(yǎng)細胞以及內皮細胞的生物學功能,并最終引起PE發(fā)病的相關機制。方法:(1)通過IHC的SP法檢測早孕期以及晚孕期胎盤組織中Gn T-V的表達水平、定位的情況。(2)采用WB和q RT-PCR研究Normal組和PE組Gn T-V的表達的變化。(3)通過慢病毒sh RNA轉染,下調人滋養(yǎng)細胞株(HTR8/Svneo細胞)、臍靜脈內皮細胞株(HUVECs)和早孕期絨毛外植體中Gn T-V的表達,并采用q RT-PCR、WB檢測Gn T-V sh RNA的干擾效率,并使用FCM檢測細胞的凋亡,MTT法檢測細胞增殖情況。(4)通過Transwell法的遷移和侵襲實驗下調了Gn-V后HTR8/SVneo、HUVECs細胞生物學功能的改變。(5)采用管腔成型實驗檢測Gn T-V是否影響HUVEC形成管樣結構的潛能。(6)利用原代模型(絨毛外植體),評估Gn T-V對絨毛外滋養(yǎng)細胞的外生性遷移影響。(7)收集絨毛外植體和兩種細胞的培養(yǎng)上清液,使用明膠酶譜法檢測其中的MMP2/9活性;WB檢測TIMP1/2的蛋白表達水平。(8)對HTR8/SVneo、HUVECs進行缺氧/復氧(H/R)處理,了解其對細胞生物學功能以及Gn T-V的表達情況的影響。(9)最后將細胞置于缺氧/復氧,的情況下,通過干擾Gn T-V基因及阻斷其下游FAK-ERK信號通路觀察細胞功能是否發(fā)生改變,從而了解Gn T-V調控細胞的功能在胎盤的發(fā)生和在PE發(fā)病中的作用和影響。結果:(1)通過IHC,我們發(fā)現Gn T-V在正常胎盤組織(早孕和晚孕)及PE組織中均有表達,主要定位于CTBs、STBs、TC中和血管內皮細胞(ECs)的胞漿內。而Gn T-V在蛻膜組織中,主要表達在EVTs和蛻膜細胞(DCs)中。且Gn T-V在PE組的表達較Normal組有升高的趨勢。(2)在HTR8/Svneo細胞和HUVECs中干擾Gn T-V的表達,可促進前者的遷移和侵襲能力和后者的遷移和管樣結構成型的潛能。(3)通過對HTR8/Svneo細胞以及HUVECs缺氧/復氧處理,可導致其功能減弱,伴隨著Gn T-V表達上升。而我們還發(fā)現H/R處理使得了兩個細胞中的FAK-ERK通路發(fā)生了明顯活化。(5)使用sh RNA干擾Gn T-V基因表達和ERK1/2特異性的抑制劑PD98059阻斷FAK-ERK通路,都對H/R所致的細胞功能受損有明顯的改善。結論:(1)Gn T-V參與對滋養(yǎng)細胞和內皮細胞的細胞功能的調控,可能在整個妊娠期間都發(fā)揮著作用。(2)下調Gn T-V的表達促進HTR8/Svneo細胞的侵襲和遷移能力,促進HUVECs遷移及管樣結構成型的潛能。(3)在氧化應激的損傷下,Gn T-V的表達上升,可通過調控FAK-ERK信號通路減弱滋養(yǎng)細胞的侵襲力和內皮細胞的管樣結構成型的潛能,從而參與PE的發(fā)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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R714.244

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