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研究生:鄭采倩
研究生(外文):Tsai-Chien Cheng
論文名稱:研究Eps8參與在EGFR調控細胞生長訊息傳遞的影響
論文名稱(外文):Study of Eps8 in EGFR-mediated mitogenesis and signal transduction
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:2005
畢業學年度:93
語文別:中文
論文頁數:85
中文關鍵詞:細胞生長訊息傳遞
外文關鍵詞:Eps8EGFRsignal transduction
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  Eps8(EGFR receptor pathway substrate NO.8)是EGFR以及non-receptor tyrosine kinase例如:Src 的一個共同受質,在許多細胞中具有97-kDa及68-kDa兩個isoforms。先前研究發現,大量表達EGF receptor的細胞以EGF刺激時,可以看到Eps8上tyrosine磷酸化明顯增加。同時,在EGF recptor大量表達的細胞內送入Eps8,會促進EGF所產生的mitogenesis與transformation。

  然而我們觀察到:在EGFR overexpressing cells已經伴隨有Eps8 overexpression的情形。為了瞭解Eps8的確參與在EGFR促進生長及transformation的signaling pathway,我們將表達Eps8 siRNA的DNA plasmid 送入EGFR overexpressing cells,觀察在EGF刺激下,其對於細胞的生長及致癌能力有何影響。

  首先,我們挑出表現eps8 siRNA (eps8 siRNA-12與siRNA-16)及p97eps8-specific siRNA (p97eps8 siRNA-5與p97eps8 siRNA-9)的EGFR overexpressing cells (NeoR細胞株),以及表現negative control siRNA (ctrl-3與ctrl-5)的細胞株;我們發現,不論是表現eps8 siRNA或p97eps8-specific siRNA的細胞內Eps8的表達均受到抑制,且細胞的生長上亦呈遲緩。

  更進一步地,我們以MTT cell proliferation assay和BrdU incorporation assay來觀察Eps8對EGF刺激細胞生長的影響;我們看到Eps8 knockdown的NeoR cells的細胞生長速度較control cells慢;同時EGFR所調控的total tyrosine磷酸化也受到了抑制,並影響了下游主要調控生長蛋白質包括ERK和AKT的活化。但是,Shc的蛋白質表現量和磷酸化情形卻反而增加,而且和EGFR之間的interaction也較control cells為多。
 Eps8 (EGF receptor pathway substrate NO.8) is a common substrate for both EGF receptor (EGFR) tyrosine kinase and cytoplasmic tyrosine kinase Src. It exists in two isoforms p97EPS8 and p68Eps8 in many cell lines. Early studies have indicated that ectopical overexpression of p97Eps8 in EGFR overexpressing cells not only enhances its mitogenic responsiveness to epidermal growth factor, but also elevates EGF-dependent cellular transformation.

 However, we observed that Eps8 expression is elevated in EGFR-overexpressing C3H10T1/2 fibroblast cells (NeoR). Thus, whether Eps8 really participates in EGFR-mediated mitogenesis and transformation becomes an interesting issue.

 To address this, we generated eps8 siRNA and p97eps8-specific siRNA - overexpressing cells from NeoR cells. Interestingly, the EGF-induced cell growth is abolished in both p97eps8siRNA- and eps8 siRNA-overexpressing cells. Thus, Eps8 is indeed involved in EGF-mediated mitogenesis. In addition, we examined the activity of ERK and PI3K within these EGF-stimulated cells and found that as compared with control cells, both ERK and AKT activation was down-regulated in Eps8 knockdown cells. Unexpectedly, the protein expression and Tyr317 phosphorylation of Shc was up-regulated and led to increased interaction between EGFR and Shc.
頁數
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 26
第二節實驗方法 29
第三章實驗結果 45
第四章討論 55
第五章圖表 62
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