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研究生:鄭立欣
研究生(外文):Li-Hsin Cheng
論文名稱:貼附及懸浮癌細胞表面纖連蛋白組裝之影響因子
論文名稱(外文):Effects of Fibronectin Assembly on Adherent and Suspended Cancer Cell Surfaces
指導教授:許垤棋鄭宏祺鄭宏祺引用關係
指導教授(外文):Dey-Chyi SheuHung-Chyi Cheng
口試委員:許垤棋鄭宏祺
口試委員(外文):Dey-Chyi SheuHung-Chyi Cheng
口試日期:2014-07-30
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程學系(所)
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:48
中文關鍵詞:懸浮癌細胞組裝纖連蛋白
外文關鍵詞:FibronectinSuspended tumor cellAssembly
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癌症轉移往往是病患死亡的主要原因,目前已知懸浮癌細胞表面纖連蛋白組裝與肺臟血管內皮細胞的CD26結合會導致轉移,因此抑制纖連蛋白組裝將有助對抗癌症轉移。已知纖連蛋白在原位癌中是扮演抑制癌細胞轉移的角色,在懸浮細胞中卻是促進癌症轉移。因此我們若能增加原位癌細胞纖連蛋白的表達同時抑制懸浮癌細胞表面纖連蛋白組裝量,可以同時抑制癌症轉移。而過去實驗室研究發現在大鼠乳癌細胞 MTF7 中RhoA activity 會調控纖連蛋白的組裝,而文獻中也得知 GTP-RhoA 會藉著促進 actin stress fiber 增加貼附型纖連蛋白的組裝,另一篇文獻指出 PI3K 會抑制 actin stress fiber,因此我們好奇 PI3K 是否藉著影響 actin stress fiber 來抑制貼附型纖連蛋白的組裝。我們抑制 PI3K 的表現觀察貼附型纖連蛋白的組裝,發現貼附細胞中纖連蛋白的組裝明顯增加,表示被 actin stress fiber 影響的纖連蛋白確實受 PI3K 所調控。因為 PI3K 是臨床常見的 target,臨床也常使用 PI3K 抑制劑來治療癌症,因此我們有興趣在細胞貼附時藉由抑制 PI3K來增加 FN達到抑制轉移的效果,但在懸浮情況下是否可行呢?我們使用 PI3KCA 之 construct 看出 PI3K 確實促進 懸浮癌細胞 FN 之表達。而使用PI3K 之抑制劑 LY294002 與懸浮癌細胞作用後發現懸浮癌細胞表面纖連蛋白明顯被抑制,符合我們的期待。未來我們將朝此方向繼續研究,找出可以阻斷癌症轉移的方法。
Cancer metastasis is the leading causes of death worldwide. Previous studies in our lab have demonstrated that the assembly of pericellular FN on cancer cells surface promotes adhesion and metastasis via binding to CD26, a surface marker found on lung epithelium. Besides its role in facilitating blood-borne cancer cells metastasis, it has been reported that FN acts as tumor suppressor in primary tumor site. By differentially regulating FN assembly on cancer cells surface, we can treat cancer more effectively. We previously found that RhoA activity regulates FN assembly in adherent MTF7. It has been well documented that GTP-RhoA plays an important role in promoting the assembly of actin stress fiber and FN matrix. On the other hand, PI3K is able to inhibit the formation of actin stress fiber. PI3K has been used as a drug target in human cancer therapy. We, therefore, hypothesized that PI3K negatively regulates FN assembly via the inhibition of actin stress fiber. Addition of PI3K inhibitor showed increased level of FN assembly in adherent cancer cells which suggests that PI3K is involved in actin-dependent FN matrix assembly. On the contrary, FN assembly was significantly increased in suspended cancer cells overexpressed constitutively active PI3K. Furthermore, FN assembly was decreased in suspended cancer cells treated with PI3K inhibitor LY294002. By understanding the differential regulation mechanisms of FN assembly by PI3K, we might be able to prevent the growth and spread of cancer cells.
第一章 緒論
1.1 研究背景與動機 1
1.2 研究目的與問題 1,2

第二章 文獻探討
2.1 纖連蛋白 3
2.2 纖連蛋白與癌症發展 4
2.3 纖連蛋白與 RhoA 4,5
2.4 纖連蛋白與 PI3K 5,6

第三章 研究設計
3.1 實驗材料與方法 7
3.1.1 實驗細胞株 7
3.1.2 細胞繼代培養 7
3.1.3 細胞表面纖連蛋白免疫螢光染色 8
3.2 研究流程 12

第四章 實驗結果
4.1 貼附大鼠乳癌細胞 MTF7 中GTP-RhoA 參與 actin stress fiber 正調控纖連蛋白組裝 13
4.2 貼附纖連蛋白組裝受到 PI3K 及 actin stress fiber 之路徑調控 13,14
4.3 PI3k促進懸浮癌細胞表面纖連蛋白組裝 14
4.4 PI3k抑制劑 LY294002 抑制懸浮大鼠乳癌細胞 MTF7 之表面纖連蛋白組裝 15

第五章 結果與討論
5.1 結論16
5.2 後續研究與建議 16,17

第六章 參考文獻 18,19
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