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系統識別號 U0026-0812200911424510
論文名稱(中文) Discoidin domain receptor 1 藉由降低 a2b1 integrin所調控的Cdc42活性抑制膠原蛋白促進之細胞伸展
論文名稱(英文) Discoidin domain receptor 1 inhibits collagen-induced cell spreading via suppression of Cdc42 activation mediated by a2b1 integrin
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 93
學期 2
出版年 94
研究生(中文) 葉儀君
研究生(英文) Yi-June Yeh
學號 s3692407
學位類別 碩士
語文別 中文
論文頁數 50頁
口試委員 口試委員-陳鴻震
指導教授-湯銘哲
召集委員-沈孟儒
中文關鍵字 膠原蛋白  細胞伸展 
英文關鍵字 Cdc42  cell spreading  integrin  Discoidin domain receptor 
學科別分類
中文摘要   Discoidin domain receptor (DDR) 是膠原蛋白的接受器。在實驗室過去的研究證實表現過量的DDR1在狗的腎臟上皮細胞株 (MDCK) 會抑制膠原蛋白所促使的細胞伸展,相反的當過量表現dominant negative DDR1則會促使細胞在膠原蛋白刺激下的伸展。細胞的伸展對於細胞的爬行是一個重要的特性,然而DDR1如何抑制細胞伸展的機制並不清楚。細胞骨架的形成影響細胞的伸展,而Rho family-GTPases是主要的調控者。為了證實Rho family-GTPases是否參與在膠原蛋白所調控的細胞伸展,我們利用pull-down assay以及transient transfection送入持續活化態或不活化態的 GTPases置MDCK細胞中,接著觀察他們是否改變膠原蛋白所促使的細胞伸展。實驗的結果顯示DDR1會降低Rac1及Cdc42的活性,但是不影響RhoA的活性。無論是持續活化態或不活化態的Rac1都不影響DDR1所抑制的細胞伸展。然而持續活化態的Cdc42恢復DDR1所抑制的細胞伸展,並且不活化態的Cdc42則抑制表現dominant negative DDR1細胞株在膠原蛋白上的伸展。這些結果顯示DDR1藉由抑制Cdc42的活性進而抑制了細胞的伸展。藉由5E8抑制a2b1 integrin的功能,我們進一步證實膠原蛋白所促使的Cdc42活化,是透過a2b1 integrin。此外FRNK細胞株也完全抑制了膠原蛋白所促使之Cdc42活性,但是DDR1並不影響FAK磷酸化的程度。總結來說,Discoidin domain receptor 1 抑制膠原蛋白促使之細胞伸展藉由Cdc42活性,而a2b1 integrin則會促使Cdc42的活性。
英文摘要  Discoidin domain receptor (DDR) is a receptor tyrosine kinase for collagen. We previously showed that over-expression of DDR1 in MDCK cells prevented cell spreading, whereas dominant negative DDR1 induced cell spreading on collagen gel-coated dish. Cell spreading is an important characteristic for cell migration, but the mechanism whereby DDR1-inhibited cell spreading is still unidentified. Cell spreading involves organization of actin cytoskeleton, which is mainly regulated by Rho family-GTPases. In order to examine whether Rho family-GTPases are involved in collagen gel-regulated cell spreading, we employed pull-down assay and transient transfection of constitutive active or dominant negative GTPases in MDCK cells and observed whether they could alter collagen-induced cell morphological changes. The results showed DDR1 decreased the activation of Rac1 and Cdc42, but had no effects on RhoA activity. Neither constitutive active nor dominant negative Rac1 could alter DDR1-inhibited cell spreading. However, constitutive active Cdc42 rescued the DDR1-inhibited cell spreading and dominant negative Cdc42 inhibited cell spreading in cells overexpressing dominant negative DDR1. These results indicate that DDR1-inhibited cell spreading is mediated by inactivation of Cdc42. With the use of 5E8, a potent a2b1 integrin blocking antibody, we found that collagen-induced activation of Cdc42 is mediated by a2b1 integrin. Furthermore, FRNK completely blocked collagen-induced decrease in Cdc42 activity, but DDR1 did not influence the phosphorylation levels of FAK. Taken together, DDR1 inhibits collagen-induced cell spreading by suppression of Cdc42 activation, which is mediated by a2b1 integrin through FAK.
論文目次 誌謝 ---------------------------------------------------------------------- 1
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Abstract ------------------------------------------------------------------ 4
中文摘要 ------------------------------------------------------------------ 5
Introduction -------------------------------------------------------------- 7
Materials and methods ----------------------------------------------------- 10
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References ---------------------------------------------------------------- 27
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作著簡歷 ------------------------------------------------------------------ 50
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