論文名稱:

綠茶多酚類化合物對人類端粒脢調控之探討

 

The modulation of telomerase expression by green tea polyphenols

研究生:

黎萬君  Wan-Chun Li

 

(以作者名查詢陽明大學館藏系統)

 

(以作者名查詢全國圖書書目資訊網)

指導教授:

林姝君  Shu-Chun Lin

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

            學號:

38917004

          學年度:

90

          語文別:

中文

          出版年:

91

關鍵字:

綠茶多酚類化合物  Green tea polyphenols

 

人類端粒脢反轉錄酵素  hTERT

 

分泌性鹼性磷酸脢  SEAP

 

胞毒殺能力  cytotoxicity

全文說明:

電子全文

        論文頁數:

74

摘要:

茶是世界上普遍性僅次於水的飲料。許多研究指出:綠茶所含之多酚類化合物對於癌症的預防及治療具有相當顯著的效果;而端粒脢的活化,在腫瘤生成及細胞不朽化(immortalization)的過程中是非常重要的步驟;綜合上列兩點:綠茶多酚類化合物是否會抑制端粒脢的表現,是一個非常值得探討的問題。本實驗中,先比較四種綠茶多酚類化合物(-)-epigallocatechin-3-gallate (EGCG)(-)-epigallocatechin (EGC)(-)-epicatechin-3-gallate (ECG)(-)-epicatechin (EC)對不同分化程度及組織來源之癌細胞株之細胞毒性。結果發現:在160 μMEGCGEGC處理下對甲狀腺癌、肝癌有80%~90%的細胞毒殺能力,而在口腔癌及肺癌細胞株中其效果更劇,約在40 μM即有70%~90%的毒殺效果;比較分化程度不同的甲狀腺癌或肝癌細胞株對EGCGEGC的細胞耐受度,發現分化程度較高的耐受度比低度分化之細胞株來的好;而正常人類口腔上皮細胞在160 μM處理時仍無顯著之毒殺效果,因此EGCGEGC對癌細胞有專一性的毒殺效果。基於端粒脢活性主要決定於人類端粒脢反轉錄酵素(hTERT)之表現,我們利用一個由hTERT啟動子(promoter)所調控的報告基因系統,觀察上述之綠茶多酚類化合物對hTERT promoter的調節作用。結果發現:處理EGCG (15-40 μM)EGC (25-40 μM)hTERT啟動子會有顯著的抑制效果,而另外兩種多酚類化合物ECGEC則無顯著的抑制現象。除此之外,定量RT-PCR偵測EGCGEGC對於內生性hTERT基因有抑制效果,亦支持上述利用外送系統所觀察到的結果。然而,EGCGEGC對端粒脢酵素活性的抑制作用,在72小時的時間區段內均無法觀察到。綜合實驗之結果,本研究證明綠茶多酚類化合物對腫瘤細胞株具特異的毒殺能力,且能夠抑制腫瘤細胞株內hTERT啟動子及mRNA的量。這為綠茶之多酚類化合物對於癌症的預防及治療的效果,提出了可能的機轉。

   
   
 

Tea is one of the most widely consumed beverages, second to water. Many researches pointed out that the polyphenols in green tea had significantly preventive and inhibitory effects for tumorigenesis. A critical molecular event in nearly all cancer cells is the high activity of telomerase that extents telomere and directs the acquisition of immortalization phenotype. In this study, we assessed the possibility that potential green tea chemopreventive polyphenol compounds (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin- 3-gallate (ECG) and (-)-epicatechin (EC) exert their function through specific cytotoxicity to cancer cells and regulating the expression of hTERT, the catalytic subunit of telomerase. First, we detected the difference of cytotoxicity among various cancer cell lines and normal cells. By treating hepatoma cells and thyroid cancer cells with 160 µM EGCG or EGC, the major tea polyphenol compounds, up to 80~90% cells were killed. Oral and lung cancer cell lines were even more sensitive to EGCG and EGC that 40 µM of these drugs were sufficient to kill 70~90% cells. No cytotoxicity was observed when cells were treated with ECG or EC. Normal cells were resistant to all four polyphenols suggesting a role of tumor-specific cytotoxicity for EGCG and EGC. Regarding the relationship between differentiation states and cytotoxicity, we found well-differentiated cells were more resistant to EGCG and EGC than poorly- differentiated ones. Secondly, we used an hTERT promoter controlled reporter system to screen the influence of abovementioned chemicals on the promoter activity of hTERT. EGCG (15-40 µM) and EGC (25-40 µM) repressed the hTERT promoter activity in a dose- and time-dependent manner. No repressive activity on hTERT promoter by EC and ECG was found. In addition, quantitative RT-PCR confirmed the repression of endogenous hTERT mRNA levels by EGCG and EGC. However, no repression of telomerase activity by EGCG or EGC was observed in up to 72 hr. In conclusion, we found that EGCG and EGC had tumor-specific cytotoxicity and they were also inhibitiors for hTERT promoter. These results suggest possible mechanisms for the green tea polyphenols in their ability in inhibiting tumorigenesis.