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研究生:翟維君
研究生(外文):Wei-Chun Chai
論文名稱:日本腦炎病毒藉由與V-ATPase有關的內噬作用進入蚊蟲細胞之研究
論文名稱(外文):A Study on the entry of Japanese encephalitis virus into mosquito cells through V-ATPase-dependent endocytosis
指導教授:陳維鈞陳維鈞引用關係
指導教授(外文):Wei-June Chen
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:63
中文關鍵詞:內噬作用細胞質細胞膜專一性表現量
外文關鍵詞:Relationsfamily
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  • 被引用被引用:1
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日本腦炎病毒為黃質病毒屬其中的一員,此病毒經由內噬作用進入哺乳類動物細胞已經有初步的確立,但對於其如何進入蚊子細胞卻還沒有很明確的答案。 雖然病毒感染蚊子細胞的情形會被V-ATPase的專一性抑制劑所干擾,但在電子顯微鏡的觀察下又發現在中性pH質的條件下,病毒會和細胞膜直接融合而把基因體釋放到細胞質內。
為了釐清日本腦炎病毒是藉由何種機制進入蚊子細胞,我們將蚊子細胞株 ─ C6/36細胞感染日本腦炎病毒,發現細胞出現了酸性小泡表現量上升的情形,更甚者造成小泡之所以會呈現酸性的酵素 ─ vacuolar(H+)-ATPase(V-ATPase)在蛋白質以及mRNA的層面都有增加的趨勢,由此可以知道病毒進入蚊子細胞是經由細胞內噬作用。 內噬作用現今比較闡明的是與clahrin或是與微囊(caveolae)相關,利用不同的內噬作用專一性抑制劑,來干擾日本腦炎病毒進入蚊子細胞,可以知道病毒所用的細胞內噬作用是與clathrin相關。
由這些結果顯示,日本腦炎病毒進入蚊蟲細胞是藉由一種會表現V-ATPase、且與clathrin相關的內噬作用,與病毒進入哺乳類動物細胞相似。
Japanese encephalitis virus (JEV) is a member of the family Flaviviridae. It has been reported that JEV infection of C6/36 mosquito cells was inhibited by bafilomycin A1, a specific inhibitor of vacuolar type H+-ATPase (V-ATPase). However, electron microscopic studies have demonstrated that JE and type 2 dengue viruses may directly penetrate into C6/36 mosquito cells at physiological pH. It is not generally accepted that direct penetration of virions is the mode of the flavivirus entry in the productive infection. There have been reports that flaviviruses enter the cell by receptor-mediated endocytosis, primarily in mammalian cells. Especially, clathrin-dependent and caveola-dependent pathways were two main pathways in the virus entry mechanism. Endocytic activity of cells can be analysed by using pharmacological agents. We analysed the effects of chlorpromazine on JEV production and its relations with V-ATPase in C6/36 cells. The results showed that acidification of intracellular compartments was dependent on V-ATPase activity in the cells. When JEV infected C6/36 cells, the expression of V-ATPase was raised up at both protein and mRNA levels. The JEV titer in chlorpromazine-treated C6/36 cells was lower than that in untreated cells.
These reports demonstrated that JEV was taken up by the cells through a constitutive endocytic pathway, and low pH in the vesicles, presumably regulated by V-ATPase, plays an important role in intracellular translocation of the virions. The inhibition of virus infection in C6/36 cells by chlorpromazine also demonstrated that endocytosis of JEV by mosquito cells is clathrin-dependent.
目錄
授權書…………………………………………………..……………….iii
致謝……………………………………………………………...………vi
目錄…………………………………………………………….………..ix
圖表目錄………………………………………………………………..xii
中文摘要………………………………………………….………...….xiii
英文摘要…………………………..………….……….………….……xiv
第一章 文獻資料回顧………...………………………………….….…..1
第一節 日本腦炎病毒……………….………………….…….…1第二節:病毒進入細胞的機制……………………….…………6
第二章 研究設計……………………………………….………………11
第三章 實驗方法………………………………….……………………13
第一節 材料……………..………………………………….…..13
一:細胞株….…………………………………….…………….13
二:病毒株…………………….………………….…………….13
第二節 實驗方法……………………..…………….…………..13
一:細胞繼代培養………….……………………………….….13
二:病毒培養……….……………………………………….….14
三:病毒定量………………….………………….…………….14
四:病毒感染細胞後病毒效價測試…….…………….……….15
五:V-ATPase多株抗體(polyclonal antibody)製備量...............16
六:免疫螢光抗體試驗…………………………….…………..16
七、ECL西方墨漬分析法………………………….…………..17
八:反轉錄-聚合酶鏈反應……………….…….…….……......23
九:吖啶橙染色試驗………………..………….…..….…....… 24
十:Chlorpromazine (CPZ)處理C6/36細胞濃度測試…..…....25
十一:Chlorpromazine (CPZ) 及 Filipin III 處理試驗…..…..26
十二:即時定量聚合酶連鎖反應…………………….………..26
第四章 結果……..…..……………………………………….…………29
第一節 利用吖啶橙觀察日本腦炎病毒感染C6/36細胞後對細胞的影響…..……………………………………….……………29
第二節 日本腦炎病毒感染C6/36細胞後對細胞內V-ATPase的影響…………………………………………….………………..30
第三節 日本腦炎病毒感染C6/36細胞後對其V-ATPase基因表現量的影響…………..………...…………….….………………31
第四節 即時定量聚合酶連鎖反應分析日本腦炎病毒感染C6/36細胞後V-ATPase基因表現量…………………………...32
第五節 日本腦炎病毒感染C6/36細胞後病毒效價之變化…..34
第六節Chlorpromazine對日本腦炎病毒感染C6/36細胞的影響………………………………………………………….……..34
第七節Filipin III對日本腦炎病毒感染C6/36細胞的影響..…35
第五章 討論………………………………………………………….…37
文獻參考………………………………………………………….……..44

圖目錄
圖一:C6/36細胞感染日本腦炎病毒之吖啶橙染色.....……………….52
圖二:圖一C6/36細胞感染日本腦炎病毒之吖啶橙染色量化結果…53
圖三:日本腦炎病毒感染C6/36細胞後的V-ATPase及病毒免疫螢光染色..……………………………..…………………………….……….54
圖四:日本腦炎病毒感染C6/36細胞不同時間後的V-ATPase表現量……………………………………………………………………..…55
圖五:Bafilomycin A1處理C6/36細胞後,感染日本腦炎病毒之吖啶橙染色……………………….…………………………….………...….56
圖六:C6/36細胞感染日本腦炎病毒後,以反轉錄-聚合酶鏈反應方式偵測V-ATPase mRVA的表現.....…………………………………...….57
圖七:C6/36細胞感染日本腦炎病毒後,以即時定量聚合酶連鎖反應方式偵測V-ATPase mRVA的表現………………………………….…58
圖八:日本腦炎病毒感染C6/36細胞後病毒效價變化情形........…….59
圖九:Chlorpromazine (CPZ)處理C6/36細胞濃度測試………………60
圖十:Cholorpromazine影響日本腦炎病毒對C6/36細胞的產出性感染.....………………………..……………………………………..…….62
圖十一:Filipin III影響日本腦炎病毒對C6/36細胞的產出性感染…63
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