本文選題：上皮間質(zhì)轉(zhuǎn)化 + 足細(xì)胞��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：在腎臟,足細(xì)胞附著在腎小球基底膜外,其足突上多種分子相互交錯(cuò)形成裂孔隔膜(slit diaphragm,SD),是腎小球?yàn)V過(guò)屏障的重要組成部分。足細(xì)胞在保護(hù)腎小球?yàn)V過(guò)屏障的完整性方面起著重要的作用。足細(xì)胞的功能與腎臟功能密切相關(guān),其超微結(jié)構(gòu)的變化或與功能相關(guān)的分子表達(dá)改變均與腎臟功能的損害有著密切的關(guān)系。一般來(lái)說(shuō),上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)是極化上皮細(xì)胞發(fā)生許多生化變化,以獲得間充質(zhì)細(xì)胞表型。足細(xì)胞足上皮細(xì)胞,當(dāng)受到不同的刺激(如TGF-β,高濃度的葡萄糖,血管緊張索Ⅱ,阿霉素等)時(shí),有可發(fā)生EMT。當(dāng)EMT的發(fā)生,上皮表型標(biāo)志物如E-cadherin,P-cadherin,nephrin、podocalyxin,Synaptopodin 的下調(diào),與間質(zhì)表型標(biāo)志物如desmin,FSP-1,MMP-9,ILK,fibronectin,vimentin,α-SMA,Ⅰ 型膠原及 snai 等上調(diào)。在受損的腎臟產(chǎn)生腎間質(zhì)纖維化中,EMT被認(rèn)為是一個(gè)關(guān)鍵因素。足細(xì)胞EMT可引起腎小球?yàn)V過(guò)屏障的損傷和蛋白尿,嚴(yán)重者可導(dǎo)致足細(xì)胞由腎小球基底膜脫離和細(xì)胞凋亡,導(dǎo)致足細(xì)胞數(shù)量減少,從而加重蛋白尿和腎臟功能損傷甚至腎小球硬化。足細(xì)胞EMT是研究蛋白尿和腎臟纖維化發(fā)生發(fā)展的一個(gè)重要方面。研究目的:探討在高濃度的葡萄糖和血管緊張素Ⅱ存在下,足細(xì)胞的EMT以及TCF8在足細(xì)胞EMT中的影響。方法:足細(xì)胞經(jīng)分化后,經(jīng)與血管緊張素Ⅱ和葡萄糖分別孵育,Western blotting方法檢測(cè)蛋白表達(dá);Transwell小室檢測(cè)細(xì)胞的遷移和侵襲能力。qPCR方法檢測(cè)mRNA表達(dá)。結(jié)果:1.血管緊張素Ⅱ和高糖對(duì)正常足細(xì)胞E-cadherin、α-catenin、N-cadherin和vimentin蛋白表達(dá)的影響血管緊張素Ⅱ和葡萄糖可使足細(xì)胞上皮標(biāo)志分子E-cadherin和α-catenin蛋白的表達(dá)逐漸降低,而足細(xì)胞間質(zhì)標(biāo)志分子N-cadherin和Vimentin表達(dá)均升高。2.血管緊張素Ⅱ和高糖對(duì)正常足細(xì)胞TCF8蛋白表達(dá)的影響足細(xì)胞經(jīng)葡萄糖或血管緊張素Ⅱ孵育后,隨著葡萄糖或血管緊張素Ⅱ濃度的增加,TCF8蛋白的表達(dá)逐漸升高。3.血管緊張素Ⅱ和高糖對(duì)正常足細(xì)胞遷移和侵襲能力足細(xì)胞經(jīng)葡萄糖或血管緊張素Ⅱ作用后,其遷移和侵襲能力有明顯增加。4.TCF8 siRNA 篩選足細(xì)胞經(jīng)TCF8基因沉默后,TCF8的表達(dá)明顯降低,其中1#siRNA的作用最強(qiáng)。5.沉默 TCF8 基因?qū)?E-cadherin、a-catenin、N-cadherin 和 vimentin 蛋白表達(dá)的影響未經(jīng)轉(zhuǎn)染的細(xì)胞上皮標(biāo)志物分子E-cadherin和α-catenin均有表達(dá),而間質(zhì)標(biāo)志物分子N-cadherin和vimentin較低。經(jīng)高糖或血管緊張素Ⅱ刺激后,E-cadherin 和 α-catenin 表達(dá)降低,N-cadherin 和 vimentin 表達(dá)增加。shTCF8#1轉(zhuǎn)染細(xì)胞后,高濃度葡萄糖和血管緊張素Ⅱ均可使上皮細(xì)胞標(biāo)志物E-cadherin和α-catenin表達(dá)增加,間質(zhì)標(biāo)志物分子N-cadherin和vimentin表達(dá)降低,表明TCF8表達(dá)沉默后,細(xì)胞不利于間質(zhì)轉(zhuǎn)換,提示TCF8 具有促進(jìn)EMT作用。6.沉默TCF8基因?qū)ψ慵?xì)胞遷移和侵襲能力未經(jīng)轉(zhuǎn)染的細(xì)胞經(jīng)高糖或血管緊張素Ⅱ刺激后,細(xì)胞遷移和侵襲能力增加,TCF8基因沉默后,細(xì)胞遷移和侵襲能力降低。表明TCF8表達(dá)沉默后,細(xì)胞遷移能力下降。結(jié)論:本研究結(jié)果提示高糖和血管緊張素Ⅱ可促進(jìn)足細(xì)胞EMT,TCF8促進(jìn)了該EMT過(guò)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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R692

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