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研究生:詹博淵
研究生(外文):Po-Yuan Chan
論文名稱:綠茶中的多酚類物質-EGCG提升肝癌細胞株對5-FU治療時的敏感性
論文名稱(外文):Green Tea Compound EGCG Sensitizes Hepatoma Cell Lines to 5-Fluorouracil Treatment
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:生化與生醫工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:83
中文關鍵詞:綠茶素5-氟化尿嘧啶表皮生長因子接受器細胞凋亡
外文關鍵詞:EGCG5-FUEGFRapoptosis
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人類肝癌是一種對化療具有抗性且預後不佳的腫瘤,即使應用一些新興的養生療法所呈現的效果也是有限;表沒食子兒茶素-3-沒食子酸酯((-)-Epigallocatechin-3-gallate,EGCG)是綠茶中主要的多酚類物質,研究顯示其能抑制多種癌症細胞株生長且能抑制表皮生長因子受器的活性。根據本實驗結果顯示,EGCG能加強5-氟化尿嘧啶(5-FU)對於SK-Hep-1、Hep G2細胞株治療的敏感性;實驗中我們結合EGCG和5-FU來觀察肝癌細胞的存活率、DAPI、Annexin V 染色分析、細胞週期、和EGFR磷酸化在肝癌細胞的表現;發現EGCG與5-FU聯合使用具有協同作用的效果;比起單獨使用5-FU,聯合EGCG能使細胞週期停止在G1期、及誘發細胞凋亡的發生;除此之外更能抑制表皮生長因子所誘導的表皮生長因子受器的磷酸化;因此根據這些結果顯示在肝癌細胞表皮生長因子受器扮演著重要的角色;將表皮生長因子受器抑制劑與胸苷酸合成酶化療藥物結合對於治療癌症可能成為一種新興有效的治療途徑。
Hepatocellular carcinoma is at the forefront of chemotherapy -resistant tumor with poor prognosis. Even with innovative treatment regimens, response rates remain low and the duration of response is short. (-)-Epigallocatechin-3-gallate (EGCG), the principal polyphenol in green tea, has been shown to inhibit the growth of many cancer cell lines and to suppress the activity of epidermal growth factor receptor (EGFR). In this report, we examined whether EGCG was capable of enhancing the sensitivity of human hepatoma cell lines SK-Hep-1 and Hep G2 to 5-fluorouracil (5-FU) treatment. We examined the cell viability,apoptotic body formatiion,DAPI, Annexin V,cell cycle and EGFR phosphorylation in hepatoma cells using combined treatment of EGCG and 5-FU. We found EGCG was able to increase the killing effect of 5-FU via synergistic rather than additive manner. EGCG further enhanced cell arrest at G1 phase and induced apoptosis in greater degree than with 5-FU alone. Furthermore, combined use of EGCG and 5-FU inhibit efficiently EGF-induced phosphorylation of EGFR. These findings underline the fact that inhibition of EGFR may play a key role in chemoresistance of hepatoma cells and the combination of EGFR inhibiton with thymidylate synthase chemotherapy could be a novel and highly effective tool for future targeted therapy of chemoresistant tumors.
第一章 緒論------------------------------------------------------------------1
第一節、 認識肝癌--------------------------------------------------------2
一、肝癌的治療---------------------------------------------------2
二、肝癌治療上的問題------------------------------------------5
第二節、5-Fluorouracil 的介紹------------------------------------------7
一、5-Fluorouracil 的合成與特性------------------------------7
二、5-Fluorouracil的作用機制----------------------------------7
第三節、Epigallocatechin-3-gallate(EGCG) 的介紹-----------------8
一、 EGCG 的特性----------------------------------------------8
二、 EGCG 的功能----------------------------------------------9
第四節、Epidermal growth factor receptor(EGFR)與癌細胞相關性--------------------------------------------------------------------11
一、 EGFR的介紹------------------------------------------------11
二、EGFR與肝癌的關係-----------------------------------------12
第五節、細胞週期--------------------------------------------------------13
一、細胞週期的介紹---------------------------------------------13
二、細胞週期的調控---------------------------------------------14
第六節、細胞凋亡的介紹-----------------------------------------------16
一、 細胞凋亡的特性------------------------------------------16
二、 細胞凋亡的研究法---------------------------------------17
三、 細胞凋亡的機制------------------------------------------18
第七節、實驗目的--------------------------------------------------------19
第二章 材料與方法--------------------------------------------------------20
第一節、 實驗試劑------------------------------------------------------20
第二節、 細胞株的培養------------------------------------------------20
第三節、 藥物處理細胞------------------------------------------------21
第四節、 細胞存活率分析---------------------------------------------21
一、 細胞計數法-------------------------------------------------21
二、 MTT assay---------------------------------------------------22
第五節、 細胞型態觀察------------------------------------------------22
第六節、 人類血球細胞的萃取---------------------------------------22
一、 人類白血球之分離----------------------------------------22
二、 人類PBMC之分離-----------------------------------------23
第七節、 西方轉漬法---------------------------------------------------23
一、 細胞蛋白質萃取-------------------------------------------23
二、 蛋白質濃度定量-------------------------------------------24
三、 蛋白質電泳-------------------------------------------------24
四、 蛋白質轉漬-------------------------------------------------24
五、 抗體偵測----------------------------------------------------25
第八節、 細胞週期----------------------------------------------------26
第九節、 Apoptosis 的測定-----------------------------------------26
一、 Annexin-V-PI analysis-------------------------------------26
二、 Caspase activity assay-------------------------------------27
三、 細胞內分子之螢光染色----------------------------------27
第三章 實驗結果------------------------------------------------------------28
第一節、5-FU、EGCG單獨使用對於人類肝癌細胞株生長影響-28
第二節、5-FU和EGCG聯合使用-------------------------------------28
一、 細胞株生長影響---------------------------------------------28
二、 細胞型態之觀察---------------------------------------------30
三、 人類肝癌細胞EGFR的表現-------------------------------30
四、 細胞週期------------------------------------------------------31
五、 細胞凋亡相關酵素之測定---------------------------------32
第四章 討論-----------------------------------------------------------------35
第五章 結論-----------------------------------------------------------------41
參考文獻--------------------------------------------------------------------------76















圖表目錄
圖一、臺灣地區主要癌症死亡率趨勢圖-----------------------------------42
圖二、5-FU結構式--------------------------------------------------------------43
圖三、綠茶中的多酚類結構--------------------------------------------------43
圖四、Apoptosis主要路徑圖---------------------------------------------------44
圖五、EGFR家族----------------------------------------------------------------45
圖六、腫瘤中EGFR訊息傳遞路徑-----------------------------------------46
圖七、5-FU、EGCG影響人類肝癌細胞株Sk-Hep-1細胞生長-------47
圖八、5-FU、EGCG影響人類肝癌細胞株Hep G2細胞生長---------48
圖九、不同時間和濃度下EGCG和5-FU對於Sk-Hep 1細胞之--------------------------------------------------------------------------49
圖十、不同時間和濃度下EGCG和5-FU對於Hep G2細胞之影響--------------------------------------------------------------------------50
圖十一、不同濃度下EGCG和5-FU對於WBC之影響---------------------51
圖十二、不同濃度下EGCG和5-FU對於PBMC之影響-------------------52
圖十三、不同濃度下EGCG和5-FU對於Jurkat T之影響-----------------53
圖十四、不同濃度下EGCG和5-FU對於K562之影響---------------------54
圖十五、單獨和聯合使用5-FU及EGCG對於Sk-Hep 1細胞型態的影響--------------------------------------------------------------------------55
圖十六、單獨和聯合使用5-FU及EGCG對於Hep G2細胞型態的影響--------------------------------------------------------------------------56
圖十七、EGCG 250μM對於肝癌細胞株Sk-Hep 1 EGFR磷酸化之影響--------------------------------------------------------------------------57
圖十八、EGCG 500μM對於肝癌細胞株Sk-Hep 1 EGFR磷酸化之影響--------------------------------------------------------------------------58
圖十九、EGCG 250μM對於肝癌細胞株Hep G2 EGFR磷酸化之影響--------------------------------------------------------------------------59
圖二十、EGCG 500μM對於肝癌細胞株Hep G2 EGFR磷酸化之影響--------------------------------------------------------------------------60
圖二十一、5-FU、EGCG單獨及聯合使用對肝癌細胞株Sk-Hep 1 EGFR磷酸化之影響--------------------------------------------------------61
圖二十二、5-FU、EGCG單獨及聯合使用對肝癌細胞株Hep G2 EGFR磷酸化之影響--------------------------------------------------------62
圖二十三、單獨和聯合使用5-FU及EGCG導致Sk-Hep 1細胞週期的停滯-----------------------------------------------------------------------63
圖二十四、單獨和聯合使用5-FU及EGCG導致Hep G2細胞週期的停滯--------------------------------------------------------------------------64
圖二十五、藥物單獨及合併對於Sk-Hep 1細胞株P53蛋白表現之影響--------------------------------------------------------------------------65
圖二十六、藥物單獨及合併對於Hep G2細胞株P53蛋白表現之影響--------------------------------------------------------------------------66
圖二十七、5-FU、EGCG對於Sk-Hep 1細胞株caspase-3活性之影響--67
圖二十八、5-FU、EGCG對於Hep G2細胞株caspase-3活性之影響---68
圖二十九、藥物合併使用後對於Sk-Hep 1、Hep G2細胞株caspase-3活性之影響-----------------------------------------------------------69
圖三十、DAPI細胞染色在螢光顯微鏡(400X)下觀察Sk-Hep 1型態-----------------------------------------------------------------------70
圖三十一、DAPI細胞染色在螢光顯微鏡(400X)下觀察Hep G2型態-71
圖三十二、Annexin V細胞染色在光學和螢光顯微鏡下之Sk-Hep --------------------------------------------------------------------------72
圖三十三、Annexin V細胞染色在光學和螢光顯微鏡下之Hep G2型態--------------------------------------------------------------------------73
圖三十四、流氏細胞儀分析Sk-Hep 1細胞株細胞凋亡情形------------74
圖三十五、流氏細胞儀分析Hep G2細胞株細胞凋亡情形--------------75
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