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研究生:洪國富
研究生(外文):Kaou-Fu Hung
論文名稱:綠茶多酚類對於人類端粒酶促進因子活性之探討
論文名稱(外文):The Transcriptional Regulation of the Green Tea Polyphenol on hTERT Promoter Activity
指導教授:林姝君林姝君引用關係
指導教授(外文):Shu-Chun Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:口腔生物研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:綠茶多酚類化合物人類端粒酶反轉酵素
外文關鍵詞:Green tea polyphenolhTERTNF-κBAP-1
相關次數:
  • 被引用被引用:2
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  • 下載下載:141
  • 收藏至我的研究室書目清單書目收藏:1
茶是普遍性僅次於水的飲料,而綠茶中的多酚類化合物更是被指出具有多種抑制癌症生成的機制,其中包括抑制了與細胞存活息息相關的NF-kB和AP-1這兩個轉錄因子;在本實驗室先前的研究發現, 綠茶多酚類中含量最多的(-)-epigallocatechin-3-gallate (EGCG)、(-)-epigallocatechin (EGC)可以抑制癌細胞hTERT基因的轉錄作用進而導致hTERT mRNA下降,而且在hTERT啟動子上有NF-κB和AP-1的結合位置。基於以上的發現,本論文想進一步的探討在口腔癌細胞之中EGCG、EGC是否藉由NF-κB和AP-1去調控hTERT啟動子.首先探討EGCG及EGC對於SAS、OEC-M1、SCC-25和OC3這四株口腔癌細胞株的細胞毒性為何,結果發現EGCG及EGC在SAS和OEC-M1這兩株分化程度較差的細胞株中的半致死劑量約為30 μM,然而對於SCC-25與OC3這兩株分化程度較完全的細胞株其半致死劑量約為160 μM,而進一步利用定量RT-PCR觀察hTERT mRNA在10、20、30 μM EGCG及EGC處理之下被抑制的情形,結果發現SAS與OEC-M1在處理20、 30 μM的EGCG及EGC時hTERT mRNA有顯著的下降;反之,SCC-25與OC3其hTERT mRNA則都不受影響。除此之外, SAS細胞內的NF-kB和AP-1的DNA結合活性較OEC-M1強得許多。因此選用SAS進行研究EGCG及EGC抑制hTERT基因轉錄的機制。在EMSA實驗結果發現AP-1與NF-kB在處理30 μM EGCG後6小時其DNA結合能力開始下降,在18小時活性抑制程度最大;而在EGC方面則是在1~2小時活性抑制程度最大,之後活性會慢慢的恢復,進一步則是利用染色質免疫沉澱實驗觀察,這些活性受影響的AP-1與NF-κB是否在活體內與hTERT啟動子做專一性結合,結果顯示細胞內的NF-κB和AP-1與hTERT啟動子無明顯結合。本研究證明了EGCG及EGC在較惡性癌細胞中非但可以引起較強的細胞毒性,也會抑制hTERT mRNA的表現。雖然被抑制的AP-1及NF-kB活性並不是造成hTERT mRNA 表現下降的主因。
Tea is the most widely consumed beverage, second to water. It had been demonstrated that the green tea polyphenols exhibit molecular function of cancer chemoprevention, including suppression of NF-κB and AP-1 which are closely related to cell survival. Our previous investigation has shown (-)-epigallocatechin-3-gallate (EGCG) and (-)-epigallocatechin (EGC), the most common polyphenols in the green tea polyphenols could repress hTERT mRNA expression by inhibiting the transcription of hTERT gene. Since, NF-κB and AP-1 binding motifs are present on the hTERT promoter, this thesis attempts to study further whether EGCG or EGC regulate the hTERT gene via NF-κB and AP-1 signal pathway in the oral cancer cells. Cytotoxicity of EGCG and EGC on various oral cancer cell lines including SAS、OEC-M1、OC3 and SCC-25 was determined. Up to 90% of cells were killed by 80~160 μM EGCG and EGC in SAS and OEC-M1, which are poorly differentiated cell lines. However, in the well- differentiated SCC-25 and OC3 cell lines, only 50% cells were killed even with 160 μM treatment. Real-time quantitative PCR was then used to detect the hTERT mRNA expression in SAS and OEC-M1 cells after treatment with 10、20、30 μM EGCG and EGC. The hTERT mRNA expression in SAS and OEC-M1 cells was inhibited profoundly with 20 or 30 μM EGCG and EGC treatment for 24 h . However,EGCG and EGC had no effect on the hTERT gene expression in OC3 and SCC-25 cell lines. SAS cells exhibited higher endogenous DNA binding activity of AP-1 and NF-κB than OEC-M1 cells in EMSA analysis. Therefore, SAS cells, in which the hTERT gene can be down-regulated by EGCG and EGC, were used to investigate the mechanism of hTERT transcription repression. EMSA analysis revealed that the repression of AP-1 and NF-κB binding activity by EGCG and EGC was dose dependent. With 30 μM EGCG, the inhibition appeared at 6h and reached the maximum at 18 h. With 30 μM EGC treatment, the binding activity was inhibited between 1~2 h and recovered gradually. Furthermore, chromatin immunoprecipitation(ChIP) was used to observe the specific binding of AP-1 and NF-κB to hTERT promoter in vivo. The result indicated the lack of interaction of AP-1 and NF-κB with hTERT promoter. In conclusion, EGCG and EGC can induce cancer cells death but also inhibit hTERT mRNA expression in the more advanced neoplastic cells. The repression of DNA binding activity of AP-1 and NF-κB did not seem to contribute to hTERT mRNA expression.
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