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研究生:莊雅淳
研究生(外文):Ya-Cnun Chuang
論文名稱:v-Src調控Eps8蛋白質表達的研究
論文名稱(外文):Studies of v-Src-mediated expression of Eps8
指導教授:呂增宏
指導教授(外文):Tzeng-Horng Leu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:85
中文關鍵詞:蛋白質表達
外文關鍵詞:SrcEps8
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中文摘要(Abstract in Chinese)
Eps8是上皮生長因子接受器(EGFR)的其中一個受質。在EGFR過量表現的細胞內,Eps8具有加強EGF對細胞分裂和致癌的能力。在我們實驗室先前的研究中,已經證實了Eps8不論是在細胞內或細胞外皆會被v-Src所磷酸化。而且在v-Src transformed的IV5細胞中,p97Eps8及p68Eps8這兩種同源異質蛋白質表現量都會增加,尤其在p68Eps8中這個情形更為明顯。所以,活化態的Src所誘導的Eps8酪胺酸磷酸化和蛋白質的大量表現,可能會促進細胞分裂及腫瘤形成。在本研究中,利用C3H10T1/2、v-Src轉型的細胞(IV5)及大量表現對溫度敏感的Src的細胞(LA29)這三種細胞株,我們證明了Src的kinase活性對Eps8的蛋白表現量非常重要。首先,我們分別對這三株細胞以不同的抑制劑處理,觀察v-Src所調控的Eps8蛋白質表現是透過何種途徑所調控。由結果發現,不論是在C3H10T1/2、IV5或LA29的細胞中,當處理以PP2(Src抑制劑)和TSA(HDAC抑制劑)24小時後,皆觀察到細胞內蛋白質的酪胺酸磷酸化受到抑制,及Eps8蛋白質表現量下降。再進一步以RT-PCR去觀察eps8 RNA的表現量,發現在C3H10T1/2細胞中,只有在PP2處理下eps8的RNA表現有下降的情形,表示PP2會影響eps8的轉錄作用。然而在v-Src細胞內,TSA可以抑制eps8的基因轉錄作用,造成Eps8蛋白量的下降。由於v-Src所誘導的Eps8酪胺酸磷酸化及其蛋白質的表現都會促進細胞分裂及癌化。因此,我們想了解eps8基因是如何受到調控。所以,我們要將老鼠eps8啟動子的區域選殖出來以利於對v-Src如何的調控eps8啟動子進行探討。
英文摘要(Abstract in English)
Eps8 is one of the EGF receptor substrates and its overexpression may contribute to the mitogenic and oncogenic effects of EGF receptor. Our previous studies indicated that Eps8 was phosphorylated by Src both cell free system and in vivo. In addition, the protein expression of both p97Eps8 and p68Eps8 isoforms are elevated in v-Src transformed cell (IV5). Thus, both tyrosyl phosphorylation and protein expression of Eps8 may contribute to Src-mediated mitogenesis and oncogenesis. In this study, by utilizing C3H10T1/2, v-Src transformed cells (IV5) and temperature-sensitive v-Src-overexpressing cells (LA29), we demonstrated that both Src kinase activity and protein expression are important for v-Src-mediated Eps8 expression. First, we treated C3H10T1/2, IV5 and LA29 cells in the presence of various inhibitors for 24 hrs. Inhibition of protein tyrosyl phosphorylation as well as reduced Eps8 expression were observed in all these three cell lines treated with PP2 and TSA. Furthermore, we utilized RT-PCR to check the RNA expression of eps8. We observed reduced eps8 transcript in reponse to PP2 in C3H10T1/2 cells and TSA in IV5 cells. This indicated that both PP2 and TSA could reduce eps8 transcript. Given that p97Eps8 overexpression causes cellular transformation of normal C3H10T1/2 fibroblast, understanding of how eps8 gene is regulated thus becomes an important issue. To achieve this goal, we cloned the murine eps8 promoter to dissect the mechanisms responsible for Src-mediated Eps8 expression. Further characterization of the eps8 promoter region should be able to illustrate how Src-mediated Eps8 expression in the future
目錄
中文摘要 1
英文摘要 4
縮寫檢索表 7
第一章 緒 論 10
第二章 實驗材料及方法
第一節 實驗材料 20
第二節 實驗方法 26
第三章 實驗結果 39
第四章 討 論 48
圖 表 55
參考文獻 70
參考文獻(References)
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賴俊儒 (1997). eps8在細胞生長與v-Src導致細胞轉型功能上的研究。國立成功大學藥理學研究所碩士論文。
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