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研究生:陳泰安
研究生(外文):Tai-An Chen
論文名稱:探討抗B型肝炎病毒藥物的新方法: 驗證抗糖尿病藥物, Metformin一類新型的 抗B型肝炎藥物
論文名稱(外文):A new approach to search for anti-HBV drug: to identify anti-diabetic drug, metformin, as novel class of anti-HBV therapeutics
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:2012
畢業學年度:100
語文別:中文
論文頁數:59
中文關鍵詞:抗B肝
外文關鍵詞:metfoemin
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本實驗室過去研究在肝癌細胞株Hep3B/T2中發現胰島素確實能夠抑制 B型肝炎病毒顆粒的產生。有趣的是,調控核心啟動子的活性和參與糖質新生基因的表現的轉錄因子有相似之處。此外已知胰島素與Metformin治療第II類型糖尿病是透過抑制肝臟進行糖質新生作用(gluconeogenesis),有鑑於此,提出了一個假說為Metformin是否能成為一種新穎的藥物,藉由調控宿主的糖質新生作用而影響了 B 型肝炎病毒基因的表現。首先,利用Luciferase reporter assay,發現 cAMP 及葡萄糖皮質素dexamethasone 促進核心啟動子活性及PEPCK 和 G6Pase mRNA具有協同的效果 (synergistic effect) ,再利用B 型肝炎病毒核心啟動子不同長度序列剔除 (serial deletion) 的核心啟動子以及不同區域的點突變 (point mutation),鑑定出 nt1656-1675 為核心啟動子上Metformin的反應序列 (response element)。在過去有研究報告指出,利用帶有 B 型肝炎病毒基因的轉殖鼠進行短時間禁食時,發現體內 B 型肝炎病毒及PGC-1α表現量上升,PGC-1α在維持能量的穩定上扮演著重要的角色,它可以和 HNF-4α結合去活化糖質新生 (gluconeogenesis)的相關基因。
接著進一步發現在Hep3B/T2細胞,Metformin會透過活化AMPK路徑影響PGC-1α蛋白質的表現,進而影響了B 型肝炎病毒核心啟動子與G6Pase啟動子的活性,為了更加了解Metformin活化AMPK路徑和B 型肝炎病毒基因的關聯,透過特定的抑制劑以及活化劑處理細胞做進一步探討,發現處理A769662 (AMPK活化劑)會抑制cAMP/dexamethasone 促進核心啟動子及G6Pase啟動子的活性以及抑制PGC-1α蛋白質的表現;另外處理AMPK抑制劑Compound C,當AMPK受到抑制時,Metformin 就無法抑制8-bromo-cAMP/dexamethasone 所活化G6Pase mRNA的表現,同樣地,Metformin 就無法抑制核心啟動子的活性,另外發現大量表現PGC-1α蛋白質同樣也可以回復Metformin 抑制核心啟動子的活性,有趣的是核心啟動子上nt1656-1675 的構築質體p19nt*3會受到cAMP/dexamethasone以及大量表現PGC-1α蛋白質的活化。在本論文的研究中,發現治療第II類型糖尿病Metformin具有抗HBV的效果,進一步發現Metformin抑制核心啟動子及糖質新生基因的表現是透過活化AMPK路徑,而轉錄輔因子PGC-1α也參與在其中,希望本論文的研究對於慢性肝炎以及糖尿病患者的治療能有所貢獻。

Our laboratory has found that insulin inhibited the hepatitis B virus particles generated in the hepatoma cell line Hep3B/T2. Interestingly, the regulation of core promoter activity and transcription factors of gluconeogenesis gene expression is similar. In addition, insulin and Metformin treatment of type II diabetes through inhibition of liver gluconeogenesis. In light of this, it is therefore hypothesize that anti-diabetic drug, metformin, as novel class of anti-HBV therapeutics, by regulation of host gluconeogenesis gene expression. First, it was shown that combination of cAMP and glucocorticoids had a synergistic effect on the induction of the HBV core promoter activity and PEPCK and G6Pase mRNA. Then, it was identified nt1656-1675 which as a response element of Metformin in the HBV core promoter using serial deletion as well as point mutation at different regions of the core promoter.
Previous studies have shown that peroxisome proliferators-activated receptor-γ coactivator 1 (PGC-1) is strongly induced to coactivate HBV gene expression in liver of fasting mice. PGC-1 is a major metabolic regulator of key gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) , it is associated with HNF-4α binding to activated gluconeogenesis.
In Hep3B/T2 cells, Metformin suppress hepatitis B virus core promoter and G6Pase promoter activity through inhibits PGC-1α protein expression by activated AMPK, and the inhibition was resembled with specific activator A769662 (AMPK activator) ; another treated the AMPK inhibitor compound C, when AMPK was inhibited, Metformin was not be able to inhibit the cAMP/dexamethasone activated the G6Pase mRNA. Similarly, Metformin was not be able to inhibit the activity of the core promoter when treated with compound C. On the other hand, overexpression of PGC-1α protein can reverse Metformin inhibition of the core promoter activity, it is interesting that nt1656-1675 on the core promoter construct plasmid p19nt 3 was stimulated by cAMP/dexamethasone, and overexpression of PGC-1α protein activation.
This study has revealed that treatment of cells the first type II diabetes Metformin has anti-HBV effect, and further found that Metformin inhibition of the core promoter and gluconeogenic gene expression via activation of AMPK pathway, and the transcription cofactor (co-activator) of PGC -1α is also involved in this pathway, Then it is hope of this research can contribute to the treatment of chronic hepatitis B and patients with diabetes.

中文摘要…………………………………………………….…...……………4
Abstract…………………………………………………….….………………5
壹. 緒論(Introduction) …………………………………….………………….7
1-1 B 型肝炎病毒簡介……………………………….…………..……7
1-2 B 型肝炎病毒結構……………………………….…………..……8
1-3 B 型肝炎病毒基因之表現與調控…………...……………………8
1-4 慢性B 型肝炎病毒治療之藥物及其優缺點……………………10
1-5 糖質新生作用 (gluconeogenesis) …..……………………………11
1-6 Metformin…………………………………………………………12
1-7 研究動機…………………………….………………….…………13
貳. 材料與方法 (Materials and Methods)…….…..…………..…………….14
2-1 使用儀器及設備……………………………………………………14
2-2 材料 (Materials) ……………………………………………………15
2-3細胞株 (cell line) …………………………………...………………17
2-4 細胞培養 (cell culture) ……………………..……...………………17
2-5 細胞生長分析 (Analysis of cell growth) …………..………………17
2-6 B 型肝炎病毒表面抗原量分析 (HBsAg test) ……..……………17
2-7 質體轉染 (transfection) ……………………………………………18
2-8 啟動子活性分析 (promoter activity assay) ……………….………18
2-9 β-galactosidase activity assay……………………………..………18
2-10 製備全細胞萃取物 (whole cell lysate extraction)…….…………18
2-11 蛋白質濃度測量………………………………………..…………19
2-12 西方墨點法 (Western blotting) ………….………………….……19
2-13 核醣核酸萃取 (RNA extraction) ……….………………..………19
2-14 反轉錄聚合酶鏈鎖反應 (reverse transcription-polymerase chain reaction, RT-PCR) ………………………………………………………20
2-15 即時定量聚合酶鏈鎖反應 (quantitative PCR, Q-PCR) .…..……20
2-16 病毒DNA純化 (Virus DNA Purification) ………………....……21
2-17 統計分析…………………………………………………….….…21
參. 結果 (Results) ………………………………………………..………22
3-1. Synergistic effects of 8-bromo-cAMP, and dexamethasone on HBV core promoter in Hep3B/T2 cells………………………………………22
3-2. Synergistic effects of cAMP, and dexamethasone on gluconeogenesis in Hep3B/T2 cells…………………………………………………………23
3-3. Metformin and insulin suppress gluconeogenesis and HBV core promoter activity in Hep3B/T2 cells……………………………………24
3-4. Metformin suppress PGC-1α expression through activate AMPK in Hep3B/T2 cells…………………………………………………………26
3-5. AMPK inhibitor Compound C reverses metformin suppressed core promoter activity in Hep3B/T2 cells……………………………………28
3-6. Metformin suppresses HBV replication and expression. ….………29
肆. 討論 (Discussion) ……………………………………………...………30
4-1. Synergistic effects of 8-bromo-cAMP, and dexamethasone on HBV core promoter in Hep3B/T2 cells………………………………….……30
4-2. Synergistic effects of cAMP, and dexamethasone on gluconeogenesis in Hep3B/T2 cells…………………………………………….……………31
4-3. Metformin and insulin suppress gluconeogenesis and HBV core promoter activity in Hep3B/T2 cells…………………………………….32
4-4. Metformin suppress PGC-1α expression through activate AMPK in Hep3B/T2 cells……………………………………………………….….33
4-5. AMPK inhibitor Compound C reverses metformin suppressed core promoter activity in Hep3B/T2 cells……………………………………..34
4-6. Metformin suppresses HBV replication and expression…………….35
伍.參考文獻 (References) ………………………………………….………36
陸.圖表 (Figures) …………………………………………………...………41
柒.附錄 (Appendices) ………………………………………………………55

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