Par3-aPKC与Vangl2相互作用并参与调节E-cadherin-based粘附连接和管腔化形成
发布时间：2018-04-09 11:16
本文选题：平面细胞极性 切入点：顶底细胞极性 出处：《北京协和医学院》2017年博士论文
【摘要】：目的与意义细胞极性是指因胞内物质和细胞器等的不对称分布而造成的细胞形态和结构的不对称性。上皮细胞是构成神经管、消化道等组织的一类特化的极性细胞,具有两种轴向的极性:顶底端极性(apical-basal polarity,ABP)和平面细胞极性(planar cell polarity,PCP)。这类上皮细胞在发育过程中通常形成管腔性结构。ABP通路和PCP通路均由极性蛋白复合物组成。Par3是ABP极性通路最关键的核心蛋白,其突变或缺失会造成多囊肾等管腔形态异常疾病。我们课题组既往研究发现人PARD3罕见致病突变涉及人脑颅神经管缺陷发病机制。Vangl2是PCP极性通路最关键的核心蛋白,其突变或缺失会造成神经管闭合缺陷等管腔异常。E-cadherin是介导细胞粘附连接的经典的粘附分子,也是上皮细胞极性建立和维持的基础,其分布异常会造成神经管闭合缺陷。既往关于ABP通路和PCP通路的研究多独立研究分别阐述,在对果蝇和非洲爪蟾的研究中发现两条通路中部分蛋白存在相互结合,但至今对两条通路,尤其是通路中对管腔化形成最关键的核心蛋白Par3和Vangl2间的相互作用在哺乳动物未见报道。本研究首先探索了 PCP通路核心蛋白Vangl2与ABP通路核心蛋白Par3-aPKC复合物在哺乳动物上皮细胞中是否存在相互作用关系及Par3、Vangl2对E-cadherin-based细胞间粘附连接的调控作用。进而应用体外3D培养细胞模型进一步探索了极性蛋白Vangl2、Par3、粘附分子E-cadherin的表达、分布对管腔化形成的影响。以期通过对管腔形成过程中极性蛋白分子作用的了解进一步探寻管腔化异常疾病神经管闭合缺陷等病症的发病机制。材料与方法1.首先免疫荧光法检测哺乳动物神经上皮细胞形成的管腔性结构和3D培养MDCK细胞形成的管腔性结构中Vangl2与Par3-aPKC的定位表达;利用2D培养的MDCK细胞检测Vangl2与Par3-aPKC的共定位表达情况;采用免疫共沉淀(COIP)、GST-pulldown法检测Vangl2与Par3-aPKC间相互作用;2.利用慢病毒介导RNAi技术分别沉默Par3和Vangl2的表达,筛选获得Par3、Vangl2基因沉默稳定细胞株,结合Real-timePCR、免疫荧光,蛋白印迹多种方法分析Par3敲减对Vangl2表达与定位的影响以及Vangl2敲减对Par3表达与定位的影响;3.基于E-cadherin-based细胞间粘附连接在管腔化形成中的重要性,我们针对Par3的敲减细胞模型,利用免疫荧光、COIP、Transwell实验、细胞迁移实验检测Par3、Vangl2对E-cadherin-based粘附连接的调节作用;4.针对Par3的敲减细胞模型,利用体外3D培养技术模拟管腔化形成,检测Par3、Vangl2和E-cadherin对管腔化形成的影响。结果1.Par3-aPKC与Vangl2富集表达于管腔性结构的顶端,并在哺乳动物极性上皮细胞中存在共定位表达和相互作用。2.Par3敲减不影响Vangl2蛋白及mRNA水平的表达,但可以调节Vangl2的膜定位。3.哺乳动物上皮细胞中Par3、Vangl2和E-cadherin相互结合形成蛋白复合物,并且Par3、Vangl2参与调节E-cadherin-based粘附连接的形成。4.Par3敲减引起3D培养的MDCK细胞形成异常的多囊性和顶端扩张型管腔形态,同时影响Vangl2和E-cadherin在管腔结构中的定位表达。5.在Par3敲减的细胞中过表达Par3与Vangl2可以恢复正常管腔化形成。结论1.哺乳动物极性上皮细胞中PCP通路核心蛋白Vangl2与ABP通路核心蛋白复合物Par3-aPKC存在相互作用,且Par3调控Vangl2的亚细胞定位。2.Par3敲减破坏E-cadherin-based粘附连接的形成,引起Vangl2、E-cadherin蛋白细胞膜顶端部位分布减少并影响Vangl2和E-cadherin间复合体的形成,而细胞顶端区域Par3、Vangl2、E-cadherin的减少和相互作用的破坏可能参与引起异常的管腔化形态。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q25
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