【摘要】：目的:N-乙酰氨基葡萄糖轉(zhuǎn)移酶V(N-acetylglucosaminyltransferase V,Gn T-V)是N-乙酰氨基葡萄糖轉(zhuǎn)移酶家族中的一員,它主要作用是催化N-聯(lián)接聚糖形成,調(diào)控細(xì)胞的生物學(xué)功能,參與細(xì)胞的粘附、遷移、侵襲和細(xì)胞內(nèi)信號(hào)傳導(dǎo)等過(guò)程,并發(fā)揮著重要的作用。以往關(guān)于Gn T-V的研究主要集中在與腫瘤的惡性程度的關(guān)系上;而有關(guān)于Gn T-V在人類胎盤中的研究很少。所以,本實(shí)驗(yàn)將從以下幾個(gè)方面對(duì)Gn T-V在胎盤上的作用進(jìn)行探討:(1)首先,利用組織標(biāo)本(正常早孕和晚孕胎盤,后者又包括Normal control組和PE組),檢測(cè)Gn T-V定位的情況,和在PE中Gn T-V的表達(dá)是否有變化;(2)采用來(lái)自人絨毛外滋養(yǎng)細(xì)胞的細(xì)胞株HTR8/Svneo以及,人臍靜脈內(nèi)皮細(xì)胞株HUVECs(human umbilical vein endothelial cells)來(lái)檢測(cè)Gn T-V的表達(dá),研究Gn T-V對(duì)二者細(xì)胞生物學(xué)功能的調(diào)控;(3)對(duì)HTR8/Svneo細(xì)胞以及HUVECs進(jìn)行H/R的處理,模擬PE初期的氧化應(yīng)激損傷,探究Gn T-V基因是如何通過(guò)FAK-ERK信號(hào)通路調(diào)控滋養(yǎng)細(xì)胞以及內(nèi)皮細(xì)胞的生物學(xué)功能,并最終引起PE發(fā)病的相關(guān)機(jī)制。方法:(1)通過(guò)IHC的SP法檢測(cè)早孕期以及晚孕期胎盤組織中Gn T-V的表達(dá)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(dá)的變化。(3)通過(guò)慢病毒sh RNA轉(zhuǎn)染,下調(diào)人滋養(yǎng)細(xì)胞株(HTR8/Svneo細(xì)胞)、臍靜脈內(nèi)皮細(xì)胞株(HUVECs)和早孕期絨毛外植體中Gn T-V的表達(dá),并采用q RT-PCR、WB檢測(cè)Gn T-V sh RNA的干擾效率,并使用FCM檢測(cè)細(xì)胞的凋亡,MTT法檢測(cè)細(xì)胞增殖情況。(4)通過(guò)Transwell法的遷移和侵襲實(shí)驗(yàn)下調(diào)了Gn-V后HTR8/SVneo、HUVECs細(xì)胞生物學(xué)功能的改變。(5)采用管腔成型實(shí)驗(yàn)檢測(cè)Gn T-V是否影響HUVEC形成管樣結(jié)構(gòu)的潛能。(6)利用原代模型(絨毛外植體),評(píng)估Gn T-V對(duì)絨毛外滋養(yǎng)細(xì)胞的外生性遷移影響。(7)收集絨毛外植體和兩種細(xì)胞的培養(yǎng)上清液,使用明膠酶譜法檢測(cè)其中的MMP2/9活性;WB檢測(cè)TIMP1/2的蛋白表達(dá)水平。(8)對(duì)HTR8/SVneo、HUVECs進(jìn)行缺氧/復(fù)氧(H/R)處理,了解其對(duì)細(xì)胞生物學(xué)功能以及Gn T-V的表達(dá)情況的影響。(9)最后將細(xì)胞置于缺氧/復(fù)氧,的情況下,通過(guò)干擾Gn T-V基因及阻斷其下游FAK-ERK信號(hào)通路觀察細(xì)胞功能是否發(fā)生改變,從而了解Gn T-V調(diào)控細(xì)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(jié)果:(1)通過(guò)IHC,我們發(fā)現(xiàn)Gn T-V在正常胎盤組織(早孕和晚孕)及PE組織中均有表達(dá),主要定位于CTBs、STBs、TC中和血管內(nèi)皮細(xì)胞(ECs)的胞漿內(nèi)。而Gn T-V在蛻膜組織中,主要表達(dá)在EVTs和蛻膜細(xì)胞(DCs)中。且Gn T-V在PE組的表達(dá)較Normal組有升高的趨勢(shì)。(2)在HTR8/Svneo細(xì)胞和HUVECs中干擾Gn T-V的表達(dá),可促進(jìn)前者的遷移和侵襲能力和后者的遷移和管樣結(jié)構(gòu)成型的潛能。(3)通過(guò)對(duì)HTR8/Svneo細(xì)胞以及HUVECs缺氧/復(fù)氧處理,可導(dǎo)致其功能減弱,伴隨著Gn T-V表達(dá)上升。而我們還發(fā)現(xiàn)H/R處理使得了兩個(gè)細(xì)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(dá)和ERK1/2特異性的抑制劑PD98059阻斷FAK-ERK通路,都對(duì)H/R所致的細(xì)胞功能受損有明顯的改善。結(jié)論:(1)Gn T-V參與對(duì)滋養(yǎng)細(xì)胞和內(nèi)皮細(xì)胞的細(xì)胞功能的調(diào)控,可能在整個(gè)妊娠期間都發(fā)揮著作用。(2)下調(diào)Gn T-V的表達(dá)促進(jìn)HTR8/Svneo細(xì)胞的侵襲和遷移能力,促進(jìn)HUVECs遷移及管樣結(jié)構(gòu)成型的潛能。(3)在氧化應(yīng)激的損傷下,Gn T-V的表達(dá)上升,可通過(guò)調(diào)控FAK-ERK信號(hào)通路減弱滋養(yǎng)細(xì)胞的侵襲力和內(nèi)皮細(xì)胞的管樣結(jié)構(gòu)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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R714.244

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