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研究生:謝俊生
研究生(外文):Hsieh Chun Sheng
論文名稱:台灣金線連抑制肝癌細胞生長抗氧化機制的探討
論文名稱(外文):Oxidative stress mediate the anti-proliferative effect of Anoectochilus formosanus on hepatoma cells
指導教授:陳偉德陳偉德引用關係
指導教授(外文):Chen Walter
學位類別:碩士
校院名稱:中國醫藥學院
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
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:69
中文關鍵詞:台灣金線連抗氧化酵素氧化壓力
外文關鍵詞:Anoectochilus formosanusantioxidant enzymeoxidative stress
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在民間盛傳為藥虎之一的台灣金線連對保肝作用有顯著功效,本研究以台灣金線連為研究題材,試著探討台灣金線連是否具有抑制癌細胞生長的能力。以不同分化程度的肝癌細胞HepG2、Hep3B、Mal5、SK進行細胞生長抑制實驗,結果顯示在HepG2細胞株中對台灣金線連的感受性特別好,在10μg/ml濃度下即有抑制近50 %生長率的效果出現,顯示金線連對於分化程度較好的肝癌細胞有較明顯抑制生長的效果。在加藥後GSH量有顯著下降的功能,CuZn-SOD量有漸增的趨勢,catalase量在加藥後有漸少的趨勢。SOD活性隨時間而增加,catalase活性在24小時後,有相對減少的趨勢,在48小時後相對減少的量最多,在72小時後相對的減少量最少。GPx、SOD、catalase mRNA level皆有明顯之差異。luciferase activity assay結果顯示,加藥組與對照組之比較轉染具有NF-κB結合位而影響轉錄子活性約下降4.6倍;轉染具有AP-1結合位而影響轉錄子活性約下降6.1倍,兩者均有明顯之差異。推測金線連透過抑制NFκB,AP-1與GCS-HS啟動區結合造成GCS-HS mRNA合成減少,進而造成GSH表現量減少,因而使得HepG2細胞生長受到抑制。在外加過氧化氫的實驗結果顯示,以台灣金線連先處理過HepG2細胞後在1,2,4小時內對超氧自由基、過氧化氫有比較強的清償能力。
Anoectochilus formosanus is a traditional folk herb that was thought to have effect on liver function protection. Previous studies showed that rat treated with water extracts of A. formosanus had better recovery of liver injury with reduction of the AST and ALT level.
To determine the anticancer activity of A. formosanus on hepatocellular carcinoma, HepG2 cells were treated with serial concentration of water extracts of A. formosanus. Significant decrease of HepG2 cell proliferation was found with treatment of A. formosanus at 10 mg/ml.
The glutathione mRNA level and the NFκB-dependent transcriptional activity were both decreased at 24 hour after A. formosanus treatment, as revealed by the quantitative RT-PCR and luciferase reporter gene analysis.
Taken together, this study result indicated that A. formosanus may have the anticancer activity to hepatocellular carcinoma cell line and may elicit its effect through the free radical scavenger system..
中文摘要 1
英文摘要 2
謝辭 3
目錄 4
圖目錄 8
符號與縮寫
第一章 緒論 9
1.1. 台灣金線連之簡介
1.2. 台灣金線連之成分與藥理作用之研究
1.3. 自由基、活性氧分子與抗氧化酵素之介紹
1.3.1. 自由基的形成原由和導致之細胞傷害
1.3.2. 活性氧分子的種類和造成之毒性
1.4. 體內抗氧化機制
1.5 研究動機
第二章 材料與方法 20
2.1. 細胞之處理
2.2. 細胞毒性試驗
2.3. 蛋白質抽取
2.4. Glutathione assay
2.5. 以西方墨點法測定蛋白質的表現量
2.6. RNA之抽取
2.7. 以 RT-PCR 來評估基因的表現
2.8. 超氧化歧化酵素活性分析 SOD activity assay
2.9. 過氧化氫酵素活性分析catalase activity assay
2.10. 轉錄子活性分析 promoter assay
2.11. 超氧自由基分析superoxide assay
2.12. 過氧化氫分析 hydrogen peroxidase assay
2.13. 外加H2O2實驗超氧自由基、過氧化氫分析
第三章 結果 30
3.1. 台灣金線連對各種不同肝癌細胞株的生長影響
3.1.1. 台灣金線連對HepG2細胞株生長率影響之結果
3.1.2. 台灣金線連對Hep3B細胞株生長率影響之結果
3.1.3. 台灣金線連對Mal 5細胞株生長率影響之結果
3.1.4. 台灣金線連對SK細胞株生長率影響之結果
3.2. 台灣金線連對HepG2 細胞株之GSH變化量測定
3.3. 以西方墨點法測量 CuZn-SOD、 catalase 蛋白質表現情形
3.3.1 以西方墨點法分析CuZn-SOD 蛋白質表現量測定
3.3.2 以西方墨點法分析catalase 蛋白質表現量測定
3.4. 台灣金線連對HepG2 細胞株中抗氧化酵素mRNA表現
之測量
3.4.1. RT-PCR測量GCS mRNA的表現情形
3.4.2. RT-PCR測量GPx mRNA的表現情形
3.4.3. RT-PCR測量SOD mRNA的表現情形
3.4.4 RT-PCR測量catalase mRNA的表現情形
3.5. 測量 SOD、 catalase 活性
3.6. 台灣金線連對HepG2 細胞株轉錄子活性分析
3.7 台灣金線連處理HepG2 細胞株後活性氧分子產量分析
3.7.1 台灣金線連對HepG2 細胞株處理後超氧自由基產量分析
3.7.2 台灣金線連對HepG2 細胞株處理後過氧化氫產量分析
3.8 外加過氧化氫處理後HepG2 細胞株活性氧分子產量分析
3.8.1 HepG2 細胞株經外加200 μM過氧化氫處理後活性氧分子產量測定
3.8.2 HepG2 細胞株經外加200 μM過氧化氫處理後再以台灣金線連處理活性氧分子產量測定
3.8.3 HepG2 細胞株經以台灣金線連處理後再外加200 μM過氧化氫活性氧分子產量測定
第四章 討論 40
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