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研究生:劉知勉
研究生(外文):Chih-Miem Liu
論文名稱:TS95和KP-19-1-401影響人類肝癌細胞中B型肝炎病毒活性之探討
論文名稱(外文):The Effect of TS95 and KP-19-1-401 on Hepatitis B Virus in Human Hepatoma Cells
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:生物化學研究所
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:2004
畢業學年度:92
語文別:中文
論文頁數:78
中文關鍵詞:過山香五味子B 型肝炎病毒
外文關鍵詞:TS95KP-19-1-401Hepatitis B virus
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B型肝炎是一種相當普遍且常見的疾病,其帶原者往往會發展出急性或慢性肝炎,也是罹患肝癌的高危險群。目前對於B型肝炎帶原者,雖然已經發展出免疫和核酸類似物(nucleoside analogs)的治療方法,然而藥物副作用加上抗藥性的問題,發展抗B型肝炎的藥物仍然是目前迫切的議題。
近來藥物的開發,許多傳統中草藥利用細胞組織培養之體外模式,研究探討其作用機制。本論文利用含有完整B型肝炎病毒基因體的HepG2/A2細胞和部份B型肝炎病毒基因體的Hep3B/T2細胞,以抑制這兩種細胞分泌B型肝炎病毒表面抗原(HBsAg)的能力為活性指標,進行藥物篩選。本論文擬探討從植物萃取出來的純化合物,TS95和KP-19-1-401,對抑制B型肝炎病毒的調節機制。過山香在民間是用來治療蛇咬的一種中草藥,五味子在中草藥的研究中發現其具有保護肝臟組織的功能。其中TS95是從野生灌木過山香(Clausena excavate)萃取出來的純物質,KP-19-1-401是從五味子(Schizandrae Fructus)萃取出來的純物質。
隨著TS95和KP-19-1-401使用濃度的增加,Hep3B/T2細胞的B型肝炎病毒表面抗原釋出量會隨之降低;而HepG2/A2細胞的B型肝炎病毒表面抗原和套膜抗原釋出量也會隨之降低。HepG2/A2細胞在長時間處理TS95和KP-19-1-401的情況下,其表面抗原量也會隨處理時間增加而降低;在相對應的濃度處理下,對細胞的生長都沒有太大的影響。發現TS95和KP-19-1-401具有抑制B型肝炎病毒表面抗原和套膜抗原釋出量之結果。利用北方點墨法分析HepES2細胞中,B型肝炎病毒的各種RNA產物。HepES2細胞是由人類肝癌細胞株HepG2,經pHBV1.3-hygromycin轉染,以hygromycin篩選得到的穩定細胞株。HepES2細胞具有1.3倍的HBV完整基因體,能轉錄出數種不同的病毒RNA產物及複製出完整的病毒顆粒。2 □g/ml的TS95和1□g/ml□的KP-19-1-401能同時抑制HepES2細胞中3.5/3.6和2.1/2.4 kb之B型肝炎病毒RNA的表現。利用暫時性轉染的方法,將含有各種B型肝炎啟動子的質體DNA送入Hep3B/T2細胞中,以質體上luciferase基因的表現,來分析TS95和KP-19-1-401對B型肝炎四種啟動子的影響。實驗結果顯示,隨著TS95和KP-19-1-401使用的濃度增加,core和SPII啟動子的活性皆會被抑制的越低;其中KP-19-1-401對SPI啟動子也有抑制的效果,然而TS95和KP-19-1-401對X啟動子都沒有明顯的影響。
研究中同時發現,KP-19-1-401會抑制cyclin A啟動子的活性和cyclin A蛋白質的表現量,對於cyclin B1和cyclin D蛋白質則沒有影響。分析KP-19-1-401對Hep3B/T2細胞週期分佈的影響,發現1 □g/ml的KP-19-1-401會造成細胞持續性的G1 phase停滯。除了B型肝炎病毒方面的探討,KP-19-1-401對Hep3B/T2細胞所造成的影響,值得進一步的研究。
Infection by hepatitis B virus (HBV) frequently results in acute and chronic hepatitis and is associated with a high risk of developing primary hepatocellular carcinomas in human. Immunization and nucleoside analogs as drugs against HBV have been shown to be effective. Owing to the efficacy and resistance of drugs, effective drugs to eradicate HBV in chronic carriers are still not available. There is an urgent need to develop novel drug screening system to search for new anti-HBV therapeutics.
Recently, cell culture system has been used to study the mechanisms of Chinese medicinal herbs, which are widely used as a resource for the development of new drugs. Human hepatoma cell lines, HepG2/A2 and Hep3B/T2, contain integrated HBV gnomes and continually secrete HBsAg. These cell lines provide an assay system for screening of anti-HBV agents from Chinese herb medicines. This study has examined the anti-HBV activities of several pure compounds extracted from traditional Chinese herbs. Clausena excavate has been used for snake biting. Schizandrae fructus has exhibited protective effect in the liver injury. TS95 and KP-19-1-401 were extracted from Clausena excavate and Schizandrae fructus respectively.
Both TS95 and KP-19-1-401 suppressed the HBsAg production in a dose-dependent manner in Hep3B/T2 cells. In HepG2/A2 cells, these two pure compounds suppressed the HBsAg and HBeAg production both in time- and dose-dependent manners but had no obvious effect on cell growth. The cell line HepES2 is a clonal derivative of human hepatoblastoma, HepG2 in which the 1.3 copies of the entire HBV genome is stably transfected into the host genome, and in which HBV is replicated, resulting in the production of infectious virus. Northern blotting analysis was performed to study the HBV RNA transcripts. The HBV-specific transcripts both on 3.5/3.6 and 2.1/2.4 kb were reduced by 2 ug/ml TS95 and 1 ug/ml KP-19-1-401. We further investigated the regulatory mechanism by transiently transfecting various plasmids that contain a HBV promoter region and a luciferase reporter gene into Hep3B/T2 cells. The promoter activity was analyzed by luciferase assay. TS95 and KP-19-1-401 suppressed both CP-luciferase and SPII-luciferase activity in a dose-dependent manner. KP-19-1-401, but not TS95, suppressed SPI-luciferase activity in a dose-dependent manner. TS95 and KP-19-1-401 showed no obvious effect on X-luciferase activity.
Furthermore, KP-19-1-401 suppressed cyclin A-luciferase activity in a dose-dependent manner in Hep3B/T2 cells. The protein level of cyclin A but not cyclin B1 and cyclin D was reduced by 1 ug/ml KP-19-1-401-treated Hep3B/T2 cells. 1.0 ug/ml KP-19-1-401 induced the occurrence of cell cycle arrest at the G1 phase in Hep3B/T2 cells.
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