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研究生:陳韋均
研究生(外文):Wei-Chun Chen
論文名稱:登革熱病毒的時間序列影像觀測
論文名稱(外文):Time-Lapsed Visualization of Dengue Virus Infection
指導教授:兵岳忻
指導教授(外文):Yueh-Hsin Ping
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生醫光電工程研究所
學門:工程學門
學類:生醫工程學類
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:69
中文關鍵詞:登革熱病毒螢光影像粒子追蹤擴散運動
外文關鍵詞:Dengue virusfluorescent imageparticle trackingdiffusion
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近年來具時間解析以及空間解析的動態螢光顯微影像技術快速的發展,並廣泛應用於特定性生物分子的動態追蹤。我們建立時間序列螢光顯微影像系統以觀測登革熱病毒感染宿主細胞的動態。病毒在細胞內的運動可以看成微小粒子在膠體溶液中的擴散行為,我們以粒子軌跡追蹤程式與統計分析方法,探討登革熱病毒的擴散模式以及在宿主細胞內的輸送路徑。

經過病毒運動軌跡分析後,我們將登革熱病毒在宿主細胞中的運動行為大致分為三種情況:兩階段式擴散運動、均一擴散運動,以及多階段式的複雜運動,由以上不同的運動行為模式顯示出登革熱病毒在宿主細胞內活動的多元性以及複雜性。另外我們在細胞中加入chlorpromazine抑制clathrin調控的細胞內噬作用,發現70%的登革熱病毒被侷限在細胞膜上進行小規模的側向擴散運動,而無法進入細胞內部。為了探討登革熱病毒與細胞自噬作用的關聯性,我們同步觀測登革熱病毒以及自噬小體的動態螢光影像,根據實驗結果顯示登革熱病毒在感染過程中被輸送至宿主細胞的自噬小體後,進行膜融合以脫去其外套膜結構。

我們建立具有時間以及空間解析能力的螢光粒子影像追蹤平台,可以直接觀測病毒在宿主細胞內的運動行為以及病毒與細胞內胞器的交互作用,有利於了解病毒感染的生理機制以應用於抗病毒藥物或是疫苗的研發。
Recently, fluorescence microscopic techniques with temporal and spatial resolution have been rapidly progressed and widely applied for dynamic tracking of specific bio-molecules. We have established a time-lapsed fluorescence imaging system for the observation of Dengue virus (DENV) infection. Viral motion in host cells can be thought of as the diffusion of small-particle in colloid solution, we utilized a particle tracking program and statistical analysis methods to investigate the behaviors of viral diffusion and the infectious transport pathway in host cells.

After viral moving trajectory analysis, the motion behaviors of DENV have been classified into three types, including two-step diffusion, homogeneous diffusion, and complicated multiple-step motion, that show the diverse and complicated behaviors of DENV particles in host cells. Additionally, we treated cells with chlorpromazine to inhibit clathrin-mediated endocytosis, as the results, 70% of tracked DENV particles were blocked to enter host cells and confined on cell membrane with small-scale simple lateral diffusion. In order to unveil the relation between DENV and cell autophagy, we observed the dynamic images of DENV and autophagosome simultaneously, it showed that DENV could be transported to autophagosome after entering host cells, and then DENV fused with autophagosome membrane to remove its envelope structure.


The fluorescent particle image tracking platform with temporal and spatial resolution is capable of observation viral motion in host cells, and virus-cell interaction, it’s quite beneficial to understand the physiological mechanism of virus infection for the development of antivirus drug or vaccine.
摘要..................................................i
Abstract............................................iii

第一章 研究背景......................................1

第二章 研究動機與目的...............................13

第三章 實驗材料與方法...............................14

第四章 實驗結果.....................................21

第五章 討論.........................................28


參考文獻.............................................36
圖表.................................................42
附錄.................................................60
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