發(fā)布時間：2018-04-09 11:16

  本文選題：平面細胞極性　切入點：頂?shù)准毎麡O性　出處：《北京協(xié)和醫(yī)學(xué)院》2017年博士論文

【摘要】：目的與意義細胞極性是指因胞內(nèi)物質(zhì)和細胞器等的不對稱分布而造成的細胞形態(tài)和結(jié)構(gòu)的不對稱性。上皮細胞是構(gòu)成神經(jīng)管、消化道等組織的一類特化的極性細胞,具有兩種軸向的極性:頂?shù)锥藰O性(apical-basal polarity,ABP)和平面細胞極性(planar cell polarity,PCP)。這類上皮細胞在發(fā)育過程中通常形成管腔性結(jié)構(gòu)。ABP通路和PCP通路均由極性蛋白復(fù)合物組成。Par3是ABP極性通路最關(guān)鍵的核心蛋白,其突變或缺失會造成多囊腎等管腔形態(tài)異常疾病。我們課題組既往研究發(fā)現(xiàn)人PARD3罕見致病突變涉及人腦顱神經(jīng)管缺陷發(fā)病機制。Vangl2是PCP極性通路最關(guān)鍵的核心蛋白,其突變或缺失會造成神經(jīng)管閉合缺陷等管腔異常。E-cadherin是介導(dǎo)細胞粘附連接的經(jīng)典的粘附分子,也是上皮細胞極性建立和維持的基礎(chǔ),其分布異常會造成神經(jīng)管閉合缺陷。既往關(guān)于ABP通路和PCP通路的研究多獨立研究分別闡述,在對果蠅和非洲爪蟾的研究中發(fā)現(xiàn)兩條通路中部分蛋白存在相互結(jié)合,但至今對兩條通路,尤其是通路中對管腔化形成最關(guān)鍵的核心蛋白Par3和Vangl2間的相互作用在哺乳動物未見報道。本研究首先探索了 PCP通路核心蛋白Vangl2與ABP通路核心蛋白Par3-aPKC復(fù)合物在哺乳動物上皮細胞中是否存在相互作用關(guān)系及Par3、Vangl2對E-cadherin-based細胞間粘附連接的調(diào)控作用。進而應(yīng)用體外3D培養(yǎng)細胞模型進一步探索了極性蛋白Vangl2、Par3、粘附分子E-cadherin的表達、分布對管腔化形成的影響。以期通過對管腔形成過程中極性蛋白分子作用的了解進一步探尋管腔化異常疾病神經(jīng)管閉合缺陷等病癥的發(fā)病機制。材料與方法1.首先免疫熒光法檢測哺乳動物神經(jīng)上皮細胞形成的管腔性結(jié)構(gòu)和3D培養(yǎng)MDCK細胞形成的管腔性結(jié)構(gòu)中Vangl2與Par3-aPKC的定位表達;利用2D培養(yǎng)的MDCK細胞檢測Vangl2與Par3-aPKC的共定位表達情況;采用免疫共沉淀(COIP)、GST-pulldown法檢測Vangl2與Par3-aPKC間相互作用;2.利用慢病毒介導(dǎo)RNAi技術(shù)分別沉默Par3和Vangl2的表達,篩選獲得Par3、Vangl2基因沉默穩(wěn)定細胞株,結(jié)合Real-timePCR、免疫熒光,蛋白印跡多種方法分析Par3敲減對Vangl2表達與定位的影響以及Vangl2敲減對Par3表達與定位的影響;3.基于E-cadherin-based細胞間粘附連接在管腔化形成中的重要性,我們針對Par3的敲減細胞模型,利用免疫熒光、COIP、Transwell實驗、細胞遷移實驗檢測Par3、Vangl2對E-cadherin-based粘附連接的調(diào)節(jié)作用;4.針對Par3的敲減細胞模型,利用體外3D培養(yǎng)技術(shù)模擬管腔化形成,檢測Par3、Vangl2和E-cadherin對管腔化形成的影響。結(jié)果1.Par3-aPKC與Vangl2富集表達于管腔性結(jié)構(gòu)的頂端,并在哺乳動物極性上皮細胞中存在共定位表達和相互作用。2.Par3敲減不影響Vangl2蛋白及mRNA水平的表達,但可以調(diào)節(jié)Vangl2的膜定位。3.哺乳動物上皮細胞中Par3、Vangl2和E-cadherin相互結(jié)合形成蛋白復(fù)合物,并且Par3、Vangl2參與調(diào)節(jié)E-cadherin-based粘附連接的形成。4.Par3敲減引起3D培養(yǎng)的MDCK細胞形成異常的多囊性和頂端擴張型管腔形態(tài),同時影響Vangl2和E-cadherin在管腔結(jié)構(gòu)中的定位表達。5.在Par3敲減的細胞中過表達Par3與Vangl2可以恢復(fù)正常管腔化形成。結(jié)論1.哺乳動物極性上皮細胞中PCP通路核心蛋白Vangl2與ABP通路核心蛋白復(fù)合物Par3-aPKC存在相互作用,且Par3調(diào)控Vangl2的亞細胞定位。2.Par3敲減破壞E-cadherin-based粘附連接的形成,引起Vangl2、E-cadherin蛋白細胞膜頂端部位分布減少并影響Vangl2和E-cadherin間復(fù)合體的形成,而細胞頂端區(qū)域Par3、Vangl2、E-cadherin的減少和相互作用的破壞可能參與引起異常的管腔化形態(tài)。
[Abstract]:The purpose and significance of the cell polarity refers to the asymmetry of the cell morphology and structure caused by the asymmetric distribution of intracellular substances and organelles. The epithelial cells constitute the neural tube, a specialized cell polarity of digestive tract tissues, polarity has two axial polarity (top and bottom end: apical-basal polarity, ABP) and planar cell polarity (planar cell, polarity, PCP). This type of epithelial cells during development usually formation of lumen structure of.ABP pathway and PCP pathway by polarity protein complex composed of.Par3 core protein ABP polarity pathway key, the mutation or deletion will cause polycystic kidney disorders lumen morphology. Our previous study found that PARD3 rare pathogenic mutations involving human cranial neural tube defects in the pathogenesis of.Vangl2 is the core protein PCP polarity pathway key, the mutation or deletion will result in neural tube closure With defect of.E-cadherin is abnormal lumen adhesion molecule mediated cell adhesion connecting the classic, but also epithelial cell polarity establishment and maintenance of the foundation, which will cause the abnormal distribution of neural tube defects. Previous studies on the ABP and PCP pathways of independent research are discussed, in the study of Drosophila and Xenopus laevis were found in the there are some protein two pathways combined with each other, but still on the two pathways, especially in the lumen of the formation pathway of Par3 core protein and Vangl2 interactions between the key was reported in mammalian. This study first explored the PCP pathway and ABP pathway of Vangl2 core protein core protein Par3-aPKC complex interactions exist the relationship between Par3 and whether in mammalian epithelial cells, Vangl2 on E-cadherin-based cell adhesion regulation. Then the application of 3D in vitro cell culture model To further explore the polarity protein Vangl2, Par3, expression of adhesion molecule E-cadherin, impact on the distribution of lumen formation. The pathogenesis through the formation of polar molecules in the process of understanding to further explore the lumen of the lumen of abnormal neural tube closure defect disease and other diseases. The lumen of mammalian neural structure and 3D detection materials and methods 1. epithelial cells first immunofluorescence localization of Vangl2 and Par3-aPKC formed the culture structure formed by MDCK cells were expressed in MDCK cells; the detection of Vangl2 and Par3-aPKC by 2D culture co expression; by immunoprecipitation (COIP), the interaction between Par3-aPKC and GST-pulldown was used to detect Vangl2 expression in 2.; using lentivirus mediated RNAi silencing of Par3 and Vangl2 respectively, screened Par3, Vangl2 gene silencing stable cell lines, combined with Real-timePCR, immunofluorescence, Western blot analysis of various methods of Par3 knockdown on the expression of Vangl2 and the effect of location and knockdown of Vangl2 expression and localization of Par3 effect; 3. based on the importance of E-cadherin-based cell adhesion in the lumen of the formation of the US for the Par3 knockdown cell model by immunofluorescence, COIP, Transwell test, cell migration test the detection of Par3, regulation of Vangl2 connection to E-cadherin-based adhesion; 4. for Par3 knockdown cell culture model, simulation of lumen formation, using in vitro 3D detection of Par3, Vangl2 and E-cadherin to form the impact on the results of 1.Par3-aPKC and Vangl2 at the top of the lumen. The enrichment expressed in structure and lumen, in mammalian epithelial cell polarity in the presence of CO localization of.2.Par3 expression and interaction of knockdown did not affect the expression of Vangl2 protein and mRNA levels, but can regulate mammalian membrane localization of.3. Vangl2 Par3 epithelial cells, Vangl2 and E-cadherin combine to form protein complexes, Par3 and Vangl2, involved in the formation of.4.Par3 knockdown by 3D in cultured MDCK cells formed polycystic and top dilated lumen morphology abnormal regulation of E-cadherin-based adhesion connection, and affect the localization of Vangl2 and E-cadherin in the luminal structure in the expression of.5. in Par3 knockdown the expression of Par3 and Vangl2 cells could restore normal lumen formation. Interaction between the conclusion of the 1. mammalian polarity in epithelial cells of PCP core protein Vangl2 pathway and ABP pathway of Par3-aPKC core protein complexes, and subcellular localization of Par3 regulation of Vangl2 knockdown of.2.Par3 damage E-cadherin-based adhesion formation, Vangl2 induced formation of E-cadherin protein the top parts of the distribution of the cell membrane and reduce the effects of Vangl2 and E-cadherin complex, and the cell apex region Par3, Vangl2 The reduction of E-cadherin and the destruction of the interaction may be involved in the formation of abnormal cavities.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q25

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