RNA干扰沉默TCF8对高糖和血管紧张素Ⅱ致足细胞间质转换的研究

发布时间：2018-05-02 08:13

本文选题：上皮间质转化 + 足细胞　；参考：《山东大学》2017年硕士论文

【摘要】：在肾脏,足细胞附着在肾小球基底膜外,其足突上多种分子相互交错形成裂孔隔膜(slit diaphragm,SD),是肾小球滤过屏障的重要组成部分。足细胞在保护肾小球滤过屏障的完整性方面起着重要的作用。足细胞的功能与肾脏功能密切相关,其超微结构的变化或与功能相关的分子表达改变均与肾脏功能的损害有着密切的关系。一般来说,上皮-间质转化(epithelial-mesenchymal transition,EMT)是极化上皮细胞发生许多生化变化,以获得间充质细胞表型。足细胞足上皮细胞,当受到不同的刺激(如TGF-β,高浓度的葡萄糖,血管紧张索Ⅱ,阿霉素等)时,有可发生EMT。当EMT的发生,上皮表型标志物如E-cadherin,P-cadherin,nephrin、podocalyxin,Synaptopodin 的下调,与间质表型标志物如desmin,FSP-1,MMP-9,ILK,fibronectin,vimentin,α-SMA,Ⅰ 型胶原及 snai 等上调。在受损的肾脏产生肾间质纤维化中,EMT被认为是一个关键因素。足细胞EMT可引起肾小球滤过屏障的损伤和蛋白尿,严重者可导致足细胞由肾小球基底膜脱离和细胞凋亡,导致足细胞数量减少,从而加重蛋白尿和肾脏功能损伤甚至肾小球硬化。足细胞EMT是研究蛋白尿和肾脏纤维化发生发展的一个重要方面。研究目的:探讨在高浓度的葡萄糖和血管紧张素Ⅱ存在下,足细胞的EMT以及TCF8在足细胞EMT中的影响。方法:足细胞经分化后,经与血管紧张素Ⅱ和葡萄糖分别孵育,Western blotting方法检测蛋白表达;Transwell小室检测细胞的迁移和侵袭能力。qPCR方法检测mRNA表达。结果:1.血管紧张素Ⅱ和高糖对正常足细胞E-cadherin、α-catenin、N-cadherin和vimentin蛋白表达的影响血管紧张素Ⅱ和葡萄糖可使足细胞上皮标志分子E-cadherin和α-catenin蛋白的表达逐渐降低,而足细胞间质标志分子N-cadherin和Vimentin表达均升高。2.血管紧张素Ⅱ和高糖对正常足细胞TCF8蛋白表达的影响足细胞经葡萄糖或血管紧张素Ⅱ孵育后,随着葡萄糖或血管紧张素Ⅱ浓度的增加,TCF8蛋白的表达逐渐升高。3.血管紧张素Ⅱ和高糖对正常足细胞迁移和侵袭能力足细胞经葡萄糖或血管紧张素Ⅱ作用后,其迁移和侵袭能力有明显增加。4.TCF8 siRNA 筛选足细胞经TCF8基因沉默后,TCF8的表达明显降低,其中1#siRNA的作用最强。5.沉默 TCF8 基因对 E-cadherin、a-catenin、N-cadherin 和 vimentin 蛋白表达的影响未经转染的细胞上皮标志物分子E-cadherin和α-catenin均有表达,而间质标志物分子N-cadherin和vimentin较低。经高糖或血管紧张素Ⅱ刺激后,E-cadherin 和 α-catenin 表达降低,N-cadherin 和 vimentin 表达增加。shTCF8#1转染细胞后,高浓度葡萄糖和血管紧张素Ⅱ均可使上皮细胞标志物E-cadherin和α-catenin表达增加,间质标志物分子N-cadherin和vimentin表达降低,表明TCF8表达沉默后,细胞不利于间质转换,提示TCF8 具有促进EMT作用。6.沉默TCF8基因对足细胞迁移和侵袭能力未经转染的细胞经高糖或血管紧张素Ⅱ刺激后,细胞迁移和侵袭能力增加,TCF8基因沉默后,细胞迁移和侵袭能力降低。表明TCF8表达沉默后,细胞迁移能力下降。结论:本研究结果提示高糖和血管紧张素Ⅱ可促进足细胞EMT,TCF8促进了该EMT过程。
[Abstract]:In the kidneys, the podocytes are attached to the glomerular basement membrane, and a variety of molecules on the Poddar interlock to form slit diaphragm (SD). It is an important part of the glomerular filtration barrier. Podocyte plays an important role in protecting the integrity of the glomerular filtration barrier. The function of the podroblast is closely related to the function of the kidney. Changes in ultrastructure or changes in molecular expression related to function are closely related to impairment of renal function. Generally, epithelial-mesenchymal transition (EMT) is a variety of biochemical changes in the polarized epithelial cells in order to obtain the phenotype of mesenchymal cells. Stimulation (such as TGF- beta, high concentration of glucose, angiotensin II, adriamycin, etc.) can occur when EMT. occurs when EMT occurs, the epithelial phenotype markers such as E-cadherin, P-cadherin, nephrin, podocalyxin, Synaptopodin, and interstitial markers such as desmin, FSP-1, MMP-9, ILK, alpha, type I collagen and EMT is considered to be a key factor in the production of renal interstitial fibrosis in damaged kidneys. Podocyte EMT can cause damage to the glomerular filtration barrier and proteinuria. The serious person can cause the foot cell to be separated from the glomerular basement membrane and apoptosis, which leads to the decrease of the number of podons, thus aggravating the damage of proteinuria and renal function. To glomerulosclerosis. Podocyte EMT is an important aspect of the development of proteinuria and renal fibrosis. The purpose of this study is to explore the effect of EMT and TCF8 on the EMT of podocytes in the presence of high concentration of glucose and angiotensin II, and the effect of the podocyte on the foot cell EMT. The protein expression was detected by Western blotting method, and the cell migration and invasion ability of Transwell cells was detected by.QPCR method to detect mRNA expression. Results: 1. angiotensin II and high glucose affect the expression of E-cadherin, alpha -catenin, N-cadherin and vimentin protein in normal poddin, and the angiotensin II and glucose can make the foot cell epithelia standard The expression of E-cadherin and alpha -catenin protein decreased gradually, while the expression of N-cadherin and Vimentin in the stromal marker molecules increased the expression of.2. angiotensin II and high glucose on the expression of TCF8 protein in normal podocytes. After incubation of glucose or angiotensin II, the concentration of glucose or angiotensin II increased. Adding, the expression of TCF8 protein increased gradually,.3. angiotensin II and high glucose had the ability to migrate and invasiveness of normal poddcells. After the action of glucose or angiotensin II, the ability to migrate and invasiveness increased significantly in.4.TCF8 siRNA screening podthe cells after TCF8 gene silencing, and the expression of TCF8 was significantly reduced, of which 1#siRNA was most effective. The expression of E-cadherin, a-catenin, N-cadherin and vimentin protein expressed by the strong.5. silencing gene were expressed in the untransfected cell epithelial marker molecules E-cadherin and alpha -catenin, while the molecules N-cadherin and vimentin were lower in the interstitial markers. The expression of E-cadherin and alpha -catenin was reduced after the stimulation of high glucose or angiotensin II. Low, N-cadherin and vimentin expression increased.ShTCF8#1 transfected cells. High concentration of glucose and angiotensin II could increase the expression of E-cadherin and alpha -catenin in epithelial cell markers. The expression of N-cadherin and vimentin in the interstitial marker molecules decreased, indicating that after the expression of TCF8 is silent, the cells are not conducive to interstitial conversion, suggesting that TCF8 is promoted. The effect of.6. silencing of TCF8 gene on the cell migration and invasion of untransfected cells with high glucose or angiotensin II, increased cell migration and invasion ability, and decreased cell migration and invasion ability after TCF8 gene silencing. It indicated that the cell migration ability decreased after the silent TCF8 gene was silent. Conclusion: the results of this study suggest that high glucose is the result of this study. And angiotensin II can promote podocyte EMT and TCF8 promote the EMT process.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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