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研究生:王淑婷
研究生(外文):Shu-Ting Wang
論文名稱:利用RNAi探討V-ATPase和日本腦炎病毒感染蚊蟲細胞之關係
論文名稱(外文):Using RNAi to investigate the role of V-ATPase in C6/36 cells infected by Japanese encephalitis virus
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:2006
畢業學年度:94
語文別:中文
論文頁數:82
中文關鍵詞:V-ATPaseRNA干擾
外文關鍵詞:V-ATPaseRNAi
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日本腦炎(Japanese encephalitis)在亞洲許多國家,是很重要的蟲媒病毒傳染病,由日本腦炎病毒(Japanese encephalitis virus,簡稱JEV)感染造成。而日本腦炎病毒屬於Flaviviriade,傳染途徑是藉由蚊子傳播。
此病毒不僅能感染蚊子細胞,也能感染哺乳動物細胞。很多研究報告都已知道日本腦炎病毒感染哺乳動物細胞是經由receptor-mediated endocytosis的方式進入。在1998年Nawa以專一性抑制V-ATPase的bafilomycin A1處理蚊蟲細胞,發現日本腦炎病毒生長會受其限制,因而猜測日本腦炎病毒進入蚊蟲細胞,也是透過receptor-mediated endocytosis的方式。
所以此研究中,我們利用RNAi技術,抑制哺乳動物細胞(BHK-21) V-ATPase基因的表現,結果顯示siRNA確實造成V-ATPase mRNA表現量減少,也使得病毒複製受到限制,證實V-ATPase與日本腦炎病毒感染哺乳動物細胞上確實有一定的相關性。
隨後抑制蚊蟲細胞(C6/36)V-ATPase基因的表現,觀察其V-ATPase與病毒複製之間的關係,推測日本腦炎病毒進入蚊蟲細胞路徑是否與進入哺乳動物細胞相同。結果可以看出dsRNA確實造成了V-ATPase mRNA及protein表現量減少,但日本腦炎病毒複製能力僅微幅下降。
綜合結果顯示,日本腦炎病毒在感染哺乳動物細胞或是蚊蟲細胞,可能皆是藉由V-ATPase-regulated endocytosis。且利用RNAi技術干擾V-ATPase,影響endosome酸化能力,來抑制日本腦炎病毒在蚊蟲細胞內的表現與複製,此結果也提供我們一個從病媒方向控制日本腦炎病毒的遠景。
Japanese encephalitis is an important arboviral disease in Asia. It is caused by Japanese encephalitis virus (JEV) infection. The JEV is a member of the virus family Flaviviridae, which is transmitted to human through the bite of mosquitoes.
In addition to mosquito cells, JE virus also infects mammalian cells. Many researches have reported that JEV enters mammalian cells by receptor-mediated endocytosis. In 1998, Nawa treated mosquito cells with bafilomycin A1, a specific inhibitor of vacuolar type H+-ATPase (V-ATPase) and observed inhibited growth of JEV in the cells. These results indicate that JEV may also enter C6/36 cells through the endocytic pathway involving intracellular acidic compartments.
In this study, we first designed specific siRNAs to inhibit V-ATPase of mammalian cells (BHK-21). The results indicated that siRNA may reduce V-ATPase mRNA expression as well as virus replication. This study also demonstrated that the V-ATPase plays a role in the JEV infection to mammalian cells.
Subsequently, we used dsRNAs to knock down the V-ATPase expression of C6/36 cells based on the hypothesis that JEV takes the same route to enter the mosquito cells as does in mammalian cells. The results showed that the dsRNA could inhibit the expression at both mRNA and protein levels of the V-ATPase and reduce virus replication as well.
These results have revealed that JEV actually enters mosquitoe and mammalian cells by the V-ATPase-regulated endocytosis. In addition, functional to inhibition of V-ATPase by RNAi also blocks the JEV replication in mosquito cells. Results shown in this study have provided a promising way for the control of JEV infection in future.
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致謝……………………………………………………………………………………v
中文摘要 ……………………………………………………………………………vi
英文摘要 …………………………………………………………………………viii
第一章 文獻探討
第一節 日本腦炎及其流行病學…………………………………………1
第二節 日本腦炎病毒……………………………………………………3
第三節 RNA interference (RNAi)簡介 ………………………………6
第四節 V-ATPase (vacuolar H+-ATPase)……………………………10
第五節 具有套膜之病毒感染進入細胞的模式 ………………………12
第二章 研究動機與設計
第一節 研究目的 ………………………………………………………13
第二節 實驗設計 ………………………………………………………13
第三章 材料與方法
一. 實驗材料……………………………………………………………15
二. 病毒培養……………………………………………………………17
三. BHK-21細胞的V-ATPase subunit A之基因全長定序……………17
四. 基因選殖……………………………………………………………18
五. 構築V-ATPase RNAi construct……………………………………20
六. 細胞外轉錄及dsRNA的螢光標幟…………………………………22
七. 轉染作用……………………………………………………………23
八. 細胞存活率計算……………………………………………………23
九. 細胞內total RNA之萃取…………………………………………24
十. 即時聚合酶鍊反應…………………………………………………24
十一. 西方墨點法………………………………………………………25
十二. 免疫螢光抗體技術………………………………………………27
十三. 流式細胞儀分析…………………………………………………28
十四. 溶斑檢定法………………………………………………………28
十五. 吖啶橙染色試驗…………………………………………………29
第四章 結果………………………………………………………………………30
第五章 討論………………………………………………………………………40
參考文獻……………………………………………………………………………47
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