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研究生:王濬儒
研究生(外文):Chun-Ju Wang
論文名稱:EB病毒LMP1膜蛋白質調控IFP35蛋白質表現機轉之研究
論文名稱(外文):Regulation of IFP35 by Epstein-Barr Virus Latent Membrane Protein 1
指導教授:蔡錦華蔡錦華引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
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:79
中文關鍵詞:EB病毒干擾素干擾素誘導蛋白質LMP1IFP35
外文關鍵詞:EBVinterferonISGLMP1IFP35
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干擾素誘導蛋白質已被報導參與在干擾素之訊息傳導路徑之中,如抑制病毒複製、刺激MHC class II 抗原呈現分子的表現以及活化巨噬細胞、樹突細胞以及自然殺手細胞,以幫助宿主清除外來的病源體;然而,近年來研究指出,在許多腫瘤細胞內可發現干擾素誘導蛋白質表現量有明顯增加的現象,並且與干擾素之分泌並無直接的關連性。EB病毒感染了世界上超過95%的人口,為一具有致癌性的疱疹病毒,可使B細胞轉形為不朽化之淋巴母細胞株 (LCL)。以此特性作為模式系統,進行cDNA微陣列的分析後,可發現EB病毒之感染會刺激許多干擾素誘導蛋白質的表現。其中干擾素誘導蛋白質35 (IFP35) 已被報導可能具有增進細胞存活的能力,故選作進一步研究的對象,並釐清EB病毒調控IFP35表現之機轉。藉由轉染不同EB病毒基因於鼻咽癌細胞株NPC-TW01,並進行西方墨點法以及反轉錄聚合酶連鎖反應 (RT-PCR) 的分析,結果顯示EB病毒潛伏期所表現的膜蛋白質LMP1具有增加IFP35表現的能力。短期感染表現刪除突變體之LMP1慢病毒於Akata EBV(-) 細胞株後,經由西方墨點法以及即時定量聚合酶連鎖反應 (Q-PCR) 的分析,亦顯示CTAR1以及CTAR2兩功能區域是必須的。進一步以不同訊息傳導抑制劑進行處理後,顯示PI3K及NF-κB之活性對於LMP1去增進IFP35表現扮演重要的角色。最後進行IFP35的生物功能分析,胸腺嘧啶核苷標記嵌合法結果顯示降低內生性IFP35表現量會減緩LCL生長的速率,經流式細胞儀分析後亦發現sub-G1族群明顯上升,顯示IFP35可能具有促進細胞存活及抑制細胞凋亡的能力。而IFP35是否參與在與EB病毒之感染相關之惡性腫瘤,如淋巴癌及鼻咽癌的生成過程當中,是未來可以進一步探討的課題。
Interferon-induced proteins (IFPs) contribute a wild spectrum of IFN’s function, including suppression of virus replication, induction of MHC class II expression in antigen presenting cells, and activation of macrophages, dentritic cells and NK cells. However, the roles of IFN-independent IFP induction in many tumor cells are not fully characterized. Epstin-Barr virus (EBV) is an oncogenic gamma-herpesvirus that persistently infects over 95% of the human population. One of the strong oncognic potencies for EBV is its ability to immortalize human B cells into lymphoblastoid cell line (LCL). Using this model system, we would like to investigate the host proteins up-regulated during the EBV immortalization. By cDNA microarray analysis, we found that IFP35, a member of the IFP family with a molecular mass of 35 kDa, was increased following EBV infection in human CD19 positive primary B cells. In further study, we demonstrated that latent membrane protein 1 (LMP1) could up-regulate IFP35 expression via its two C-terminal activating region 1 and 2 (CTAR1 and CTAR2) domains, suggesting that PI3K and NF-κB signaling pathways may participate in LMP-1-induced IFP35 expression. Data from [3H] thymidine incorporation assay indicated that cell proliferation rate of LCL was significantly decreased while cells were treated with specific IFP35 sh-RNAs knockdown. Furthermore, results of PI staining revealed that the decline of growth rate was due to the increase of cell apoptosis. All the results suggest that IFP35 plays a critical role in cell proliferation and cell survival. Based on the highly significant association between EBV and tumor formation in lymphomas and NPC, we might provide the insight of functions of LMP1-induced IFP35 in EBV-associated malignancies.
口試委員會審定書……………………………………………………I
致謝……………………………………………………………………II
中文摘要………………………………………………………………III
英文摘要………………………………………………………………IV

序論……………………………………………………………………1
實驗目的………………………………………………………………13
材料與方法……………………………………………………………14
結果……………………………………………………………………33
討論……………………………………………………………………39
表………………………………………………………………………44
圖………………………………………………………………………47
參考文獻………………………………………………………………67
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