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研究生:朱琬立
研究生(外文):Wen-Li Chu
論文名稱:毛地黃素和訊息傳遞調控激酶抑制劑合併使用影響人類肝癌細胞存活率的協同作用
論文名稱(外文):The synergistic effects of digoxin and inhibitors of signal regulated kinases on survival rate of human hepatoma cells
指導教授:魏正舒魏正舒引用關係
指導教授(外文):Jeng-Shu Wei
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
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:63
中文關鍵詞:毛地黃素人類肝癌細胞上皮生長因子受體三羥異黃酮鈣離子螯合劑
外文關鍵詞:digoxinEGFRgenisteinBAPTA/AM
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我們證實人類肝癌細胞 Hep G2 和 Sk-Hep-1 以 0.1 至 1 nM 之毛地黃素 (digoxin) 抑制鈉鉀腺苷三磷酸酶 (Na+/K+-ATPase) 當作訊息傳遞複合物,活化通過上皮生長因子受體 (EGFR),使癌細胞增生分別達 39 % 和 29 %。加入酪胺酸激酶抑制劑的三羥異黃酮 (genistein) 有效阻斷低濃度毛地黃素活化上皮生長因子受體。以三羥異黃酮和毛地黃素聯合使用導致更低的細胞存活率、細胞型態的改變和細胞週期停滯在 G2/M 期,尤其是在 Hep G2 細胞上述聯合使用之作用更為明顯。以免疫轉漬法證實,0.1 nM 毛地黃素誘導上皮生長因子受體之酪胺酸磷酸化,在 Hep G2 細胞依時間的不同磷酸化程度隨之增加。使用螯合劑 BAPTA/AM 之實驗數據顯示,鈣離子流動與鈣離子可能參與毛地黃素誘導細胞增生訊息傳遞路徑。總結,三羥異黃酮和低於心臟衰竭治療濃度的毛地黃素聯合使用,造成 Hep G2 和 Sk-Hep-1 細胞死亡的協同作用。
We have demonstrated that in human hepatoma Hep G2 and Sk-Hep-1 cells, the Na+/ K+-ATPase complex acts as an intracellular signal transducing complex activated by 0.1-1 nM digoxin, which inhibit sufficient pumps to activate a transduction cascade via transactivation of epidermal growth factor receptor (EGFR), resulting in increased proliferation about 39 % and 29 %. Treatment of hepatoma cells with tyrosine kinase inhibitors such as genistein antagonized digoxin-induced activated EGFR. Combined treatment with genistein and digoxin induced lower cell viability, cell morphological change and cell cycle arrest at the G2/M phase, especially in Hep G2 cells. As demonstrated by immunoblotting, a modest induction of tyrosine phosphorylation of EGFR by digoxin was apparent at 0.1 nM. The intensity of the phosphorylated bands increased in a time-dependence manner in Hep G2 cells. As demonstrated by chelator BAPTA/AM, the requirement of Ca2+ mobilization and the Ca2+ as may share the signal pathways in digoxin-induced cell proliferation. In conclusion, combined use of genistein and digoxin at the concentration lower than its therapeutic range for the heart failure provides a synergistic effect on the cell death of Hep G2 and Sk-Hep-1 cells.
指導教授推薦書…………………………………………………………………………
口試委員會審定書………………………………………………………………………
國科會博碩士論文授權書………………………………………………………………iii
長庚大學博碩士紙本論文著作授權書…………………………………………………iv
誌謝………………………………………………………………………………………v
中文摘要…………………………………………………………………………………vi
英文摘要…………………………………………………………………………………vii
目錄………………………………………………………………………………………viii
圖表目錄…………………………………………………………………………………xi
縮寫表……………………………………………………………………………………xii
第一章 緒論……………………………………………………………………………1
第一節﹑肝癌的治療……………………………………………………………………1
一﹑肝癌化療藥物………………………………………………………………………1
二﹑以肝癌藥物治療產生的問題………………………………………………………2
第二節﹑Digoxin的介紹………………………………………………………………3
一﹑Digoxin的生理作用與功能………………………………………………………3
二﹑抑制Na+/K+-ATPase的功能……………………………………………………4
三﹑Digoxin的治療濃度範圍…………………………………………………………4
第三節﹑Digoxin對細胞的影響………………………………………………………5
一﹑有毒濃度Digoxin引發細胞凋亡…………………………………………………5
二﹑無毒濃度Digoxin引發訊息傳遞…………………………………………………6
第四節﹑細胞增生與EGFR的相關性……………………………………………………7
一﹑EGFR的介紹…………………………………………………………………………7
二﹑肝癌中EGFR的表現…………………………………………………………………8
三﹑EGFR抑制劑…………………………………………………………………………9
1. AG1478 (Tyrphostin)……………………………………………………………9
2. Gefitinib (Iressa,或稱ZD1839)……………………………………………9
3. Genistein…………………………………………………………………………10
四﹑藥物與EGFR抑制劑聯合使用………………………………………………………11
第五節﹑鈣離子參與訊息傳遞…………………………………………………………11
一﹑鈣離子之訊息傳遞…………………………………………………………………11
二﹑鈣離子對無毒低濃度強心劑作用的影響…………………………………………12
三﹑鈣離子螯合劑-BAPTA/AM………………………………………………………13
第六節﹑研究動機………………………………………………………………………14
第二章 材料與方法…………………………………………………………………15
第一節﹑實驗試劑………………………………………………………………………15
第二節﹑細胞株的培養…………………………………………………………………15
第三節﹑藥物處理細胞…………………………………………………………………16
第四節﹑細胞存活率分析………………………………………………………………16
一﹑細胞計數法…………………………………………………………………………16
二﹑MTT assay…………………………………………………………………………17
第五節﹑細胞型態觀察…………………………………………………………………17
第六節﹑細胞週期………………………………………………………………………17
第七節﹑西方轉漬法……………………………………………………………………18
一﹑細胞蛋白質萃取……………………………………………………………………18
二﹑蛋白質濃度定量……………………………………………………………………19
三﹑蛋白質電泳與轉漬…………………………………………………………………19
四﹑抗體偵測……………………………………………………………………………20
五﹑Strip………………………………………………………………………………20
第八節﹑細胞凋亡之觀察………………………………………………………………21
第三章 結果……………………………………………………………………………23
第一節﹑不同濃度Digoxin對人類肝癌細胞生長的影響……………………………23
第二節﹑無毒濃度的Digoxin促進細胞增生…………………………………………23
第三節﹑無毒濃度Digoxin活化人類肝癌細胞的EGFR………………………………24
第四節﹑無毒濃度Digoxin與酪胺酸激酶抑制劑聯合使用…………………………25
一﹑細胞存活率…………………………………………………………………………25
二﹑細胞型態之觀察……………………………………………………………………26
三﹑細胞週期……………………………………………………………………………26
四﹑細胞凋亡……………………………………………………………………………27
第五節﹑鈣離子參與無毒濃度Digoxin活化的訊息傳遞路徑………………………28
第四章 討論……………………………………………………………………………30
第一節﹑無毒濃度Digoxin促進人類肝癌細胞增生…………………………………30
第二節﹑無毒濃度Digoxin活化EGFR ………………………………………………31
第三節﹑藥物的聯合作用………………………………………………………………32
第四節﹑Genistein展現有效抑制無毒濃度Digoxin對細胞存活率的影響………32
第五節﹑證明無毒濃度Digoxin與Genistein之協同作用…………………………34
第六節﹑鈣離子對無毒濃度Digoxin促進細胞增生的影響…………………………35
第七節﹑無毒濃度Digoxin對生理上之利弊…………………………………………36
第五章 結論……………………………………………………………………………38
參考文獻…………………………………………………………………………………55
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