系統識別號 U0026-1911201220014700
論文名稱(中文) 凝血酶調節素調控上皮細胞形態與促使細胞集體爬行
論文名稱(英文) Thrombomodulin controls epithelial morphology and promotes collective cell migration
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 101
學期 1
出版年 101
研究生(中文) 許芸燕
研究生(英文) Yun-Yan Hsu
學號 S58931138
學位類別 博士
語文別 英文
論文頁數 83頁
口試委員 指導教授-施桂月
中文關鍵字 細胞-細胞附著  新生表皮  傷口癒合 
英文關鍵字 cell-cell adhesion  neoepidermis  wound healing 
中文摘要 在發育、組織更新與傷口修復的過程中,細胞間的附著作用可以維持組織的結構。凝血酶調節素(thrombomodulin,簡稱TM)是位於細胞膜的穿膜蛋白,TM藉由其細胞外的 lectin-like結構區可以媒介細胞與細胞間的連接。我們推測TM的胞內區(cytoplasmic domain)可能與細胞骨架蛋白連結,進而穩定細胞間的附著。本論文目的為研究與TM胞內區結合的蛋白質及其相關的生物功能。目前發現TM經由與ezrin結合而連接至actin細胞骨架。ezrin是屬於ezrin/radixin/moesin (ERM)家族蛋白之一,ERM蛋白已知在細胞內負責連接細胞膜蛋白與細胞骨架,與細胞型態及細胞間的附著有關。細胞外結合試驗(in vitro binding assays)指出,TM胞內區與ezrin胺基端區有直接結合。以突變分析TM胞內區確定其帶正電胺基酸(522RKK524)為主要的ezrin結合位置。免疫沉澱實驗顯示,在HaCaT與A431上皮細胞中內源性的TM與ezrin有結合。共軛焦顯微鏡分析A431細胞,結果顯示TM、ezrin以及actin細胞骨架皆存在於細胞與細胞間附著的位置。接著利用RNA干擾技術使A431細胞TM蛋白表現量降低,結果顯示降低TM的表現量會使細胞形態改變,並且促使細胞爬行。此外,使用上游的表皮生長因子刺激A431細胞,會增加TM與ezrin的結合程度。在老鼠皮膚傷口癒合的實驗,TM與ezrin會大量表現在新生的表皮組織,意謂著此兩種蛋白質是上皮再生的重要分子,上皮再生需要上皮細胞集體爬行。最後,施予外源性的TM能促使缺TM的A2058黑色素瘤細胞集體爬行。總結,本研究發現TM藉由其胞內區與ezrin結合,進而間接連接到actin細胞骨架,能調控上皮細胞形態與促使細胞集體爬行。
英文摘要 Adhesive interactions between cells are needed to maintain tissue architecture during development, tissue renewal and wound repair. Thrombomodulin (TM) is an integral membrane protein that mediates cell-cell adhesion through its extracellular lectin-like domain. We considered that the cytoplasmic domain of TM might be linked to the cytoskeleton to stabilize the intercellular adhesion. The purpose of this thesis was to investigate the interacting protein of the TM cytoplasmic domain and the relevant biological functions. It was found that TM is linked to the actin cytoskeleton via ezrin. Ezrin is a member of the ezrin/ radixin/ moesin (ERM) family of proteins that act to link membrane proteins to the actin cytoskeleton and mediate cell morphology and intercellular adhesion. In vitro binding assays showed that the TM cytoplasmic domain bound directly to the N-terminal domain of ezrin. Mutational analysis of the TM cytoplasmic domain identified positively charged amino acids 522RKK524 as important ezrin-binding residues. Immunoprecipitation experiments showed that endogenous TM interacted with ezrin in HaCaT and A431 epithelial cells. Confocal microscopy analysis of A431 cells revealed that TM colocalized with ezrin and the actin cytoskeleton at cell-cell adhesion sites. Knockdown of endogenous TM expression by RNA interference induced morphological changes and accelerated cell migration in A431 cells. Moreover, epidermal growth factor, upstream of ezrin activation, enhanced the interaction between ezrin and TM in A431 cells. In mouse skin wound healing, TM and ezrin were highly expressed in neoepidermis, implying that both proteins are key molecules in reepithelialization that requires collective cell migration of epithelial cells. Finally, exogenous expression of TM in TM-deficient A2058 melanoma cells promoted collective cell migration. In conclusion, TM, which binds to ezrin via its cytoplasmic domain to indirectly associate with the actin cytoskeleton, controls epithelial morphology and promotes collective cell migration.
論文目次 中文摘要 1
1.1 Cell-cell junctions 13
1.1.1 Tight junctions 13
1.1.2 Adherens junctions 14
1.1.3 Desmosomes 15
1.2 Thrombomodulin (TM) 15
1.2.1 Structure of TM 15
1.2.2 Expression of TM 16
1.2.3 Functions of TM 16
1.2.4 Regulation of TM expression 18
1.2.5 Soluble TM 18
1.2.6 Role of TM in tumor biology 19
1.3 Ezrin/radixin/moesin (ERM) proteins 19
1.3.1 Structure and activation of ERM proteins 20
1.3.2 Functions of ERM proteins 20
1.4 Objectives of this study 22
2.1 Magnetic isolation of cytosolic proteins bound to TM 25
2.2 Preparation of GST-TM cytoplasmic domain (TM domain 5; TMD5) full-length, truncated, and mutant fusion proteins 25
2.3 Preparation of His-tagged ezrin N-terminal domain and C-terminal domain proteins 26
2.4 GST pull-down assay 26
2.5 Solid-phase binding assay 26
2.6 Cell culture and preparation of A2058 stable cell lines 27
2.7 Immunoprecipitation and Western blotting 27
2.8 Immunofluorescence, confocal microscopy, and scanning electron microscopy 28
2.9 Preparation of TM knockdown A431 cells and ezrin knockdown cells 29
2.10 Cell migration 29
2.11 Immunohistochemistry 30
2.12 Statistical analysis 30
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