以端粒脢活性為篩選平台從當歸分離出抗腫瘤成份Butylidenephthalide並研究其藥物萃取物抗肝癌胞生長調控之基因體研究

韓鴻志
財團法人佛教慈濟綜合醫院
端粒脢主要功能是保持細胞染色體長度也和細胞老化有關，近年來發現在惡性腫瘤細胞有70% 至90% 端粒脢活性高，相對的在正常細胞只有生殖細胞及分裂的骨髓細胞才有端粒脢活性，所以以控制端粒脢活性來治療惡性腫瘤是具有專一性。至今許多證據顯示hTERT 基因控制端粒脢活性，故本實驗以 hTERT promoter+Luciferase傳入惡性肝癌細胞株 (J5) 為篩選平台，將初步分離所得萃取層加入同時傳入 (cotransfection) hTERT promoter+Luciferase及TK promoter +Renilla建構傳入於人類惡性腦瘤細胞株，隔日以測量luciferase 活性做為中藥抑制端粒脢活性之初步篩選評估。實驗結果顯示，當歸氯仿萃取層 (AS-C) (15 微克/毫升) 對人類惡性肝腫瘤 J5 細胞株的TERT 有1.5倍至5.0倍 down regulation效果，TRAP實驗也顯示AS-C對端粒脢活性抑制作用顯著，MTT 細胞毒殺實驗結果發 AS-C 可以有效地抑制肝癌細胞生長。以TUNEL 和流式細胞儀分析發現AS-C (40 微克/毫升) 使細胞環停留在G1/S且可造成明顯細胞凋零。AS-C接著以有機溶劑及不同層析法萃取，所得之純化物以質譜儀和核磁共振圖譜鑑定其分子量及結構。其中由 AS-C 萃取之純化物 (pure compound) Butylidenephthalide (MW:188.23) 對端粒脢活性有顯著抑制作用，且會引發惡性肝癌細胞凋亡，IC50 為 50 微克/毫升。為了解BP 對抗肝癌細胞之作用機轉，我們將以西方墨點法、流式細胞儀分析 和RT-PCR等技術分析BP 引起的細胞環週期和細胞凋亡情形。
關鍵字：當歸；中藥篩選平台；抗癌藥物；細胞週期；細胞凋亡；端粒脢

CCMP95-RD-211 The antitumor effect of butylidenephthalide derived from Angelica sinensis on human hepatocellular carcinoma: An example of using telomerase activity as a screening platform

Hung - Chih Han
Buddhist Tzu Chi General Hospital
Cancer cells must attain the potential for immortality to allow progression to malignant states and hence require telomerase activity. Various tumor tissues have been analyzed for telomerase activity and most are positive (>80%), but not usually detected in non-neoplastic untransformed cells. The telomerase RNA-protein complex responsible for maintenance of telomeric DNA at chromosome ends. Components of the telomeric protein complex have been cloned and the telomerase catalytic subunit has been identified and the expression of hTERT is correlated with measured telomerase activity. Therefore, the use of hTERT promoter-driven vector system is a proper model of molecular platform for drug screening. In our previous study, four varied length hTERT promoter construct was performed.
關鍵字：Telomerase；hTERT；Butylidenephthalide；Angelica sinensis；apoptosis