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研究生:林恆卉
研究生(外文):Heng-Hui Lin
論文名稱:大型D型肝炎抗原經由轉錄因子AP1調控c-fos基因
論文名稱(外文):Large hepatitis delta antigen regulates the transcription of c-fos gene through AP1
指導教授:張明富
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物化學暨分子生物學研究所
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:2008
畢業學年度:96
語文別:中文
論文頁數:55
中文關鍵詞:血清反應因子血清反應因子序列色質免疫沉澱分析微矩陣晶片D型肝炎病毒
外文關鍵詞:serum response factorserum response elementChromatin immunoprecipitation assaymicroarrayhepatitis delta virus
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D型肝炎病毒(hepatitis delta virus; HDV)是具有外套膜(enevelop)的單股負向RNA病毒,目前只發現於感染B型肝炎病人身上。HDV歸類為B型肝炎病毒的衛星病毒(satellite virus),其必須依賴B型型肝炎病毒的表面抗原組成外套膜,才能形成有感染力的病毒顆粒。HDV基因體只轉譯兩種蛋白質:小型delta 抗原(HDAg-S),含195個胺基酸(24 kDa)及 C 端多了19個胺基酸的大型delta 抗原(HDAg-L),含214個胺基酸(27 kDa),兩者在HDV病毒生活史中各有不同的任務,HDAg-S主要幫助病毒RNA複製,而HDAg-L為病毒包裹所必須。
如果B型肝炎患者重疊感染(super-infection)或共同感染(co-infection) D型肝炎病毒,會增加病情的嚴重性,例如猛爆性肝炎、慢性肝炎、肝硬化與肝癌的發生。但HDV如何加速肝癌的形成目前還不是很清楚。本實驗室先前發現大型delta抗原(HDAg-L)會活化原致癌基因c-fos的啟動子,之後,Goto等人也發現HDAg-L會活化血清反應因子(serum response factor,SRF)相關的轉錄作用,證實具有血清反應因子序列(serum response element,SRE) c-fos啟動子亦受到HDAg-L活化,但詳細的分子機轉目前還是不甚了解。
本論文的研究目主要在探討D型肝炎delta抗原對宿主細胞的影響與活化c-fos啟動子的機轉。首先利用微矩陣晶片(microarray)與Ingenuity Systems分析,發現在delta抗原存在下,HepG2細胞的mRNA表現變化主要影響許多與細胞生長及癌症相關的基因(如: TGFβ1,TGFβR2,TGFβR3,SMAD3,EGR1,EGR1,EGFR…等)。另外之研究發現在HDAg-L存在下,細胞內c-fos mRNA有增加的趨勢,為了更進一步釐清HDAg-L活化c-fos啟動子機制,目前已知SRF-Ser103磷酸化會加強及加速SRF結合至c-fos的SRE,我們發現在delta抗原存在下SRF-Ser103磷酸化有增加的現象。進一步染色質免疫沉澱分析(Chromatin immunoprecipitation assay),發現大型delta抗原存在下會減少細胞內c-Jun/c-fos結合到c-fos啟動子AP1結合之序列上,可能因而活化c-fos啟動子。
Hepatitis delta virus (HDV) is a negative sense, single-stranded RNA virus that is enveloped by the surface antigens of the hepatitis B virus (HBV). HDV is considered to be a subviral satellite because it propagates only in the presence of HBV. Inside HDV particles, HDV genomic RNA contains a closed circular RNA molecule of 1.7 kb that is associated with the viral product, delta antigen (HDAg). There are two forms of delta antigen, the small HDAg (HDAg-S, 24 kDa) and the large HDAg (HDAg-L, 27 kDa). Delta antigens are identical for the N-terminal 195 amino acids and differ only in the presence of an additional 19 amino acids at the C-terminus of HDAg-L. These two HDAgs play diverging roles in the life cycle of HDV. HDAg-S is required for the viral replication, while HDAg-L is required for the assembly of viral particles. To understand the pathogenesis of HDV, previous studies from our laboratory have demonstrated that HDAg-L could transactivate c-fos promoter. Recently, it was reported that the HDAg-L, but not HDAg-S, activated the serum response factor-dependent transcription including the transactivation of human c-fos gene. However, detailed molecular mechanisms of HDAg-L involved in the transactivation of c-fos gene through SRE and AP1 binding site are unclear. By performing microarray and Ingenuity Systems analysis in this study, expression of several host genes associated with cell growth, proliferation, and cancer were found to be affected by HDAg-L. These include TGFβ, TGFβR2, TGFβR3, SMAD3, FN1, CTNNB1, EGR1 and EGFR. In addition, realtime PCR assay showed that c-fos mRNA was upregulated in HDAg-L stable clone. Further studies demonstrated that the phosphorylation of serum response factor at Ser103 known to enhance DNA binding affinity to the serum response element of c-fos gene was increased in the HDAg-L expressing cells. In addition, chromation immunoprecipitation assay indicated that HDAg-L inhibited the binding of c-Jun to c-fos promoter in vivo.
These results suggent that HSAg-L may activate the transcription of c-fos gene by diminishing the binding of c-Jun to c-fos promoter.
中文摘要...........................................................................I
英文摘要...........................................................................II
縮寫表...............................................................................IV
緒論...................................................................................1
實驗材料來源...................................................................16
實驗方法...........................................................................19
實驗結果...........................................................................27
討論...........................................................................30
圖表...........................................................................33
參考文獻....................................................................47
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