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研究生:彭筱雯
研究生(外文):Hsiao-Wen Peng
論文名稱:TPA及胰島素是透過何種機制抑制人類B型肝炎病毒核心啟動子活性?
論文名稱(外文):How phorbol ester or insulin suppresses core promoter activity of human hepatitis B virus?
指導教授:葉小帆葉小帆引用關係
指導教授(外文):Sheau-Farn Yeh
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:70
中文關鍵詞:B型肝炎病毒胰島素核心啟動子
外文關鍵詞:hepatitis B virusTPAinsulincore promoterHNF4
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本實驗室在數年前發現TPA及胰島素在人類肝癌細胞Hep3B/T2中能有效的抑制B型肝炎病毒表面抗原 (HBsAg) 的產生,但是仍舊不清楚TPA及胰島素是否能抑制細胞中B型肝炎病毒的複製。在初步的實驗中證實TPA及胰島素能抑制1.3ES2細胞中B型肝炎病毒轉錄產物及病毒顆粒的產生。本論文將探討TPA及胰島素是如何在人類肝癌細胞中抑制B型肝炎病毒基因表現。首先在Hep3B/T2細胞中利用Luciferase reporter assay分析TPA及胰島素會影響B型肝炎病毒中的哪些啟動子來調控B型肝炎病毒基因的表現。結果顯示,在Hep3B/T2細胞中,不論TPA或胰島素皆有選擇性的抑制B型肝炎核心啟動子 (CP) 活性,但在非人類肝臟細胞如293T細胞中卻無此效果,顯示此抑制效果有肝細胞之特異性。為了解TPA或胰島素抑制B型肝炎病毒核心啟動子在Hep3B/T2細胞中之抑制機制,透過不同的MAPK抑制劑處理細胞後,發現ERK及AKT參與胰島素產生HBV核心啟動子抑制作用時訊息傳遞路徑所必需,而TPA產生抑制作用僅透過ERK訊息傳遞路徑。為確認TPA或胰島素抑制B型肝炎病毒核心啟動子的活性是透過影響核心啟動子上特定cis-element與轉錄因子的作用,利用具不同長度序列剔除(serial deletion)的核心啟動子,鑑定出Nt1656-1675為核心啟動子上TPA及胰島素的作用區段,在此區段中含有一個HNF4的結合位點。實驗亦發現TPA及胰島素能有效抑制細胞中HNF4的轉錄,在細胞中大量表現HNF4後能完全消除TPA及胰島素對B型肝炎病毒核心啟動子的抑制作用,因此最有可能的假說是TPA及胰島素是藉由抑制HNF4的表現,進而降低B型肝炎病毒核心啟動子的活性,達到抑制B型肝炎病毒轉錄及病毒顆粒的產生。
Our laboratory has shown that TPA or insulin can suppress hepatitis B virus surface antigen (HBsAg) production in human hepatoma Hep3B/T2 cells. However, whether TPA or insulin also suppresses HBV replication is still unknown. Preliminary experiments have been done to demonstrate that TPA and insulin suppress HBV transcripts and viral particle production in 1.3ES2 cells. To understand how TPA or insulin suppressing HBV gene expression in human hepatoma cells, I first examined which promoter of HBV is affected by TPA or insulin in Hep3B/T2 cells using luciferase as a reporter assay. These results clearly showed that both TPA and insulin selectively suppressed the viral core promoter (CP) activity in Hep3B/T2 cells but not in non-liver human cells such as 293T. Using selective inhibitors, we found that both ERK and AKT pathways are essential for insulin’s suppressive activity, but only ERK pathway is required for TPA’s activity. Using serial deletion mutants of core promoter, we identified Nt1656-1675 as a responsible element of insulin and TPA in the HBV core promoter. One single HNF4 binding sequence was recognized in this region. Ectopic expression of HNF4 totally abolished the suppressive activity of TPA and insulin to HBV core promoter further support the hypothesis that TPA and insulin may suppress HNF4’s expression in human liver cells to suppress HBV core promoter activity and reduce HBV transcripts and viral particle production.
中文摘要..………………………………………………………………1
Abstract…………………………………………………………………3

壹、緒論…………………………………………………………………4
1-1 B型肝炎病毒介紹…………………………………………………5
1-2 TPA及胰島素介紹…………………………………………………11

貳、材料與方法…………………………………………………………14
2-1 使用儀器及設備……………………………………………………14
2-2 材料…………………………………………………………………15
2-3 方法…………………………………………………………………19

参、結果…………………………………………………………………27
3-1 TPA及胰島素都可抑制B型肝炎病毒表面抗原產生 ……………27
3-2 TPA及胰島素可抑制細胞內B型肝炎病毒核醣核酸轉錄及病毒顆粒的產生…………………………………………………………………27
3-3 TPA、胰島素對B型肝炎病毒四個啟動子具選擇性的抑制現象…………………………………………………………………………28
3-4 TPA及胰島素對B型肝炎病毒四個啟動子活性的抑制作用具有肝組織特異性………………………………………………………………29
3-5 TPA及insulin對HBV 核心啟動子活性抑制的訊息傳遞路徑…29
3-6 鑑定B型肝炎病毒核心啟動子上TPA及胰島素的反應區段(response element) ...………………………………………………30
3-7 B型肝炎病毒核心啟動子上HNF4結合位點對 TPA及胰島素作用的影響………………………………………………………………………31
3-8 B型肝炎病毒核心啟動子上C/EBP及PPAR結合位點對 TPA及胰島素作用的影響………………………….………………………………32
3-9 TPA及胰島素藉由抑制HNF4的轉錄產物,進而影響核心啟動子的活性………………………………………………….…………………32
3-10 TPA及胰島素抑制HNF4與核心啟動子的結合能力,進而抑制核心啟動子的活性…………………………………………………………33
3-11 TPA及胰島素藉由干擾肝細胞特異性轉錄因子與核心啟動子的結合能力,進而影響B型肝炎病毒核心啟動子的活性………………33


肆、討論…………………………………………………………………35

伍、參考文獻……………………………………………………………41

陸、圖表…………………………………………………………………48

柒、附錄…………………………………………………………………63
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