臺灣博碩士論文加值系統

人類凝血酶調節素 ( Thrombomodulin, TM ) 為已知的抗凝血因子及鈣離子依賴性細胞黏附因子，於角質細胞中高度表現。我們先前研究結果顯示，靜默TM 表現會造成細胞偽足產生，進一步的蛋白質體學研究發現TM 結合蛋白包括有α- tubulin﹐β-tubulin, actin，及intersectin (ITSN)，並發現TM 藉由和ITSN 結合，調控角質細胞骨架，而細胞型態的改變會進一步的影響細胞間黏附能力，由於TM 在細胞黏附或調控細胞型態功能都類似於E-cadherin，且ITSN 已知可以調控細胞內吞噬作用，我們推測TM 可以藉由ITSN 之內吞噬作用來調控表皮黏附因子E-cadherin 的細胞胞噬及黏附功能。本論文中，靜默TM 及ITSN，發現細胞膜上E-cadherin會胞噬進入細胞質，並觀察到細胞周圍產生明顯的細胞偽足，而在鈣離子置換實驗當中，發現到靜默TM 及ITSN 細胞之E-cadherin 回復能力有降低，證實TM 及ITSN 有調控E-cadherin 的作用。最後在擬態肌膚實驗中發現， 靜默TM 及ITSN 角質細胞後，E-cadherin 表現下降，證實E-cadherin 功能有受TM 和ITSN 調控而進一步影響細胞分化的能力。由實驗結果顯示，TM 和ITSN 可以調控角質細胞的E-cadherin 的表現，並影響表皮分化能力

Thrombomodulin (TM), a calcium-dependent cell-cell adhesion molecule, is expressed in human keratinocytes and served as a differential biomarker for the clinical stages of skin cancers. Our previous study show that TM knockdown can induced lamellipodia formation, the proteomic analysis identified the interacting partners of TM including α-tubulin, β-tubulin, actin and ITSN. We also provided that TM regulats actin cytoskeleton via interaction with ITSN. Since TM is comparable to E-cadherin in adhesion and
morphoregulatory activities and ITSN is a modular scaffolding protein regulating the formation of endocytotic vesicles. We hypothesized that the TM may modulate the assembly of E-cadherin junctions by interplay with ITSN. Our results showed that si -RNA knockdown TM or ITSN increased the endocytosis of E-cadherin in HaCaT cells. The knockdown of TM or ITSN increased the lamellipodia formation whereas delayed the E-cadherin junction rebuilding by calcium switch assay. This implies that TM and ITSN play a role in forming E-cadherin-based adherens junctions. Besides, HaCaT cells downregulation of TM or ITSN were poorly differentiation by using organotypic cultures assay. These results suggest that TM and ITSN could modulate the functional of E-cadherin-mediated cell-cell adhesion and epithelial differentiation.

目錄

中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
第一章 前言 1
第一節 研究背景 1
一、 人類凝血酶調節素(Thrombomodulin; TM) 1
二、 凝血酶調節素與人類表皮角質細胞 3
三、 Intersectin (ITSN) 5
四、 細胞骨架actin之調控 6
五、 TM與ITSN 調控角質細胞actin骨架 8
六、 鈣黏著素E ( E-cadherin ) 9
第二節 研究目的 11
第二章 研究方法 12
第一節 研究設計 12
第二節 研究材料 13
一、細胞株 13
二、實驗藥劑 13
三、抗體 15
四、實驗室耗材 16
五、實驗儀器 17
第三節 實驗方法 19
一、 細胞培養 19
二、 細胞計數 20
三、 細胞繼代培養 21
四、 細胞冷凍儲存 22
五、 蛋白質萃取 22
六、 蛋白質定量 23
七、 蛋白質電泳 24
八、 西方墨點法 26
九、 免疫螢光染色 27
十、 雷射共軛焦顯微鏡 29
十一、 核糖核酸干擾技術 29
十二、 擬態皮膚培養模組 30
十三、 鈣離子置換實驗 32
第三章 研究結果 34
第一節 TM、ITSN及E-cadherin細胞內的分佈 34
第二節 靜默TM及ITSN表現影響HaCaT細胞型態 34
第三節 靜默TM或ITSN表現影響HaCaT細胞E-cadherin分佈 35
第四節 靜默TM或ITSN表現影響E-cadherin回復能力 35
第五節 靜默後的HaCaT細胞其細胞分化能力有受影響 35
第四章 討論 37
第一節 結論 39
第二節 建議 39
參考文獻 46
附錄 51

圖表目錄
圖一、TM、ITSN及E-cadherin於HaCaT細胞中的分佈 40
圖二、利用西方墨點法偵測HaCaT細胞與靜默TM、 ITSN及TM合併ITSN細胞中TM, ITSN及E-cadherin之表現量 41
圖三、HaCaT細胞與靜默TM或 ITSN後之細胞型態 42
圖四、免疫螢光染色分析HaCaT細胞及HaCaT-siRNA細胞E-cadherin之分布(A)。將胞噬之量化後分析如圖(B)。 43
圖五 calcium switch 實驗，及利用免疫螢光染色分析HaCaT細胞及HaCaT靜默TM、 ITSN﹐及TM合併ITSN 細胞中E-cadherin之分佈。 44
圖六、培養模組切片染色觀察細胞分化情形 45

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