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研究生:黃朧潁
研究生(外文):Long-Ying Huang
論文名稱:利用時間序列雙螢光顯微技術探討登革熱病毒在細胞內至自噬小體的感染路徑
論文名稱(外文):The illustration of intracellular infection route of dengue virus to the autophagosome by the time-lapsed dual fluorescent microscopy
指導教授:兵岳忻
指導教授(外文):Yueh Hson Ping
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
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:52
中文關鍵詞:登革熱病毒自噬小體時間序列雙螢光顯微技術
外文關鍵詞:Dengue virusAutophagosomeTime-lapsed dual fluorescent microscopy
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  • 被引用被引用:1
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  • 下載下載:15
  • 收藏至我的研究室書目清單書目收藏:1
登革熱病毒是現今全球分布甚廣的節肢動物媒介病毒(arbovirus)之ㄧ,屬於黃病毒屬,可透過受體媒介的胞飲作用(receptor-mediated endocytosis)進入宿主細胞,並在酸性環境中與宿主細胞進行膜融合。目前已有研究報導登革熱病毒的感染能力與自噬作用有關,然而這些研究是利用螢光免疫染色等實驗來間接證明登革熱病毒與自噬作用的關係,至於登革熱病毒如何存在自噬小體中,並在自噬小體中進行複製與轉譯作用,目前仍沒有進一步的研究提供直接的證據。由於即時影像顯微技術能在活細胞中追蹤單一病毒顆粒,因此本研究將以時間序列雙螢光影像顯微技術(Time-lapsed dual fluorescent microscopy),對登革熱病毒細胞感染之路徑作全程的觀察。本研究首先利用親脂性的螢光染劑1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt (DiD) 對病毒進行螢光標記,並利用時間序列雙螢光影像顯微技術追蹤螢光標記的登革熱病毒,發現病毒主要是利用clathrin調控的胞飲作用進入細胞,且進入細胞後,會朝細胞核方向移動。此外也發現在腎臟細胞中,登革熱病毒的感染也會激活細胞的自噬作用,而且病毒在進入宿主細胞後,脫去外套膜之前會被輸送至自噬小體,並在自噬小體中脫去外套膜。這些實驗結果暗示了登革熱病毒在進入宿主細胞後會被內體運輸至自噬小體中,之後才進行膜融合。除此之外,透過本研究說明時間序列螢光影像顯微技術為一個有力的應用工具,在未來能藉由此技術,進一步釐清病毒與宿主細胞在病毒生活史上每一個環節之間的關係。
Dengue virus (DENV) is an enveloped, positive-single strand RNA virus. It belongs to the Flavivirus which enters cells through receptor-mediated endocytosis and later fuses with host membrane in a low pH environment. Recently, observations indicated that DENV infection could induce autophagic machinery and autophagy could enhance the infectivity of DENV. However, there is no direct evidence to show how DENV transport to the autophagosome via the intracellular pathway after entering cells. Since the real-time imaging technology is a powerful tool to track the movements of a single virus in host cells, I used the time-lapsed dual-fluorescence microscopy to tackle this question. DENV particles were first labeled with a lipophilic fluorescent dye, 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt (DiD) that can anchor into DENV envelope, for visualization. Green African monkey kidney epithelial cells, Vero E6, were infected by DiD-labeled DENV particles. The infection of DiD-labeled DENV particles was monitored by the custom-built image system. The time-lapsed images revealed that DENV utilized clathrin-mediated endocytosis to enter host cells because Chlorpromazine (CPZ) treatment blocked DENV motions. Moreover, DENV infection could induce autophage in host cells, and DENV could be transported to the autophagosome before membrane fusion. These results indicate that DENV is transported to the autophagosome by early endosome and triggered membrane fusion in the amphisome. This study also suggests that time-lapsed dual fluorescent microscopy system can be a powerful tool to investigate the intracellular trafficking of virus.
誌謝 -------------------------------------------------- i
目錄 -------------------------------------------------- ii
中文摘要 ---------------------------------------------- 1
英文摘要 ---------------------------------------------- 2
研究背景 ---------------------------------------------- 3
研究目的 ---------------------------------------------- 11
實驗材料與方法 ---------------------------------------- 12
結果 -------------------------------------------------- 18
討論 -------------------------------------------------- 25
參考文獻 ---------------------------------------------- 30
圖表 -------------------------------------------------- 36
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