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研究生:蔡欣伶
研究生(外文):Shin-Ling Tsai
論文名稱:探討家禽里奧病毒誘導自體吞噬之蛋白質體學研究
論文名稱(外文):Comprehensive protein expression profiling toward ARV-induced autophagy in Vero cells
指導教授:劉宏仁 博士徐睿良 博士
指導教授(外文):Hung-Jen Liu, Ph.D.Jue-Liang Hsu, Ph.D.
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:生物科技系所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:116
中文關鍵詞:自體吞噬飢餓家禽里奧病毒感染穩定同位素標示多維液相層析-串聯式質譜
外文關鍵詞:autophagystarvationavian reovirus infectionstable-isotope labelingLC-MS/MS
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自體吞噬主要對胞質蛋白質降解,並且在胞器及長半衰期蛋白質的汰換
上扮演重要角色。細胞受到飢餓、胞器損傷、病原體入侵及氧化壓力下,
可因細胞生存或細胞防禦產生自體吞噬。由前人的研究發現家禽里奧病
毒(ARV)感染非洲綠猴腎細胞(Vero cell)可誘發自體吞噬,本研究將進一
步探討病毒感染與饑餓誘發的自體吞噬其蛋白質體之差異。利用散彈槍
蛋白質體分析的方式,使用穩定同位素標示及多維液相層析-串聯質譜
(LC-MS/MS)針對自體吞噬現象進行全面性探討。初步結果顯示,在超
過774 個鑑定出的蛋白質中,部分蛋白質受病毒所引起的自體吞噬影響,
表現量有被促進或抑制的情形。這些結果進一步與受飢餓所引起的自體
吞噬作比較。1433E、HNRPR 及S61A1 蛋白均在此二種誘發條件下有一
致的表現趨勢。FKBP3、LRC59 及RS6 則受此二種誘發條件同時抑制表
現。同時,在病毒與飢餓處理同時也鑑定到自噬小體 (phagosome)相關蛋
白質,如ATP6V1A、ACTBL、TBA4A 及RAB5B。此外,也有一些蛋白
質只受病毒感染所調控,如TCP4、S10A6 及DNPEP 等,可能可作為病
毒誘發自體吞噬之標記分子。

Autophagy is responsible for cytoplasmic bulk degradation and plays an important role for the turnover of organelles and long lived proteins. Upon cellular stress, such as starvation, organelle damage, pathogen invasion, and oxidative stress, autophagy can be induced for the purpose of cell-survival or cellular defense. In the previous study, the autophagy was observed in Vero cells upon avian reovirus (ARV) infection. In this work, the comparative proteomics between virus-induced and starvation-induced autophagy was further investigated. In order to provide proteome-wide perspective towards this phenomenon, shotgun proteomics approach coupled with stable-isotope labeling and multidimensional liquid chromatography-tandem mass
spectrometry (LC-MS/MS) were employed in this study. More than seven hundred of proteins were identified in our data, 1433E, HNRPR and S61A1 proteins were consistently regulated in ARV-infected and III starvation-stimulated Vero cells; some proteins such as FKBP3、LRC59 and RS6 were simultaneously down-regulated. Meanwhile, some phagosome
proteins such as ATP6V1A, ACTBL, TBA4A and RAB5B were also
identified from the virus and starvation-induced Vero cells. Interestingly,some proteins, such as, TCP4, S10A6 and DNPEP were regulated uniquely in ARV-infected Vero cells instead of starvation-induced ones, which may be regarded as biomarker candidates of virus-induced autophagy.

中文摘要………………………………………………………………………I
Abstract………………………………………………………………………II
謝誌…………………………………………………………………………IV
目錄…………………………………………………………V
圖目錄……………………………………………………………………VIII
表目錄………………………………………………………………………X
附表目錄……………………………………………………………………XI
第一章 序言………………………………………………………………1
第二章 文獻探討………………………………………………2
2.1 胞吞作用……………………………………………………………2
2.2 自體吞噬……………………………………………………………2
2.2.1 自體吞噬過程與步驟…………………………………………3
2.2.2 自體吞噬之分子機制…………………………………………4
2.2.3 調控自體吞噬之訊息傳遞……………………………6
2.2.4 調控自體吞噬調控因子…………………………………………7
2.3 家禽里奧病毒之歷史………………………………………………8
2.3.1 里奧病毒的基因組……………………………………………8
2.3.2 里奧病毒的外型結構…………………………………………9
2.3.3 里奧病毒的物化特性………………………………………9
2.3.4 里奧病毒的傳染途徑及症狀診斷……………………………9
2.3.5 里奧病毒的增殖特性………………………………………10
2.4 蛋白質體學…………………………………………………10
2.4.1 蛋白質體學技術……………………………………………11
2.4.1.1串聯式質譜儀 ……………………………………………11
2.4.1.2 二維液相層析-串聯式質譜分析………12
2.4.1.3 穩定同位素搭配多維液相層析-串聯式質譜分析………13
2. 4. 2 蛋白質身份鑑定……………………… ………… 14
2.5 偵測自體吞噬之方法…………………………………15-18
2.6 研究目的…………………………………………………………19
第三章 材料與方法………………………………………………………20
3.1 實驗流程簡介………………………………………………………21
3.2 藥品及設施………………………………………………………21-22
3.3 細胞培養……………………………………………………………20
3.3.1 Vero 培養與繼代……………………………………… ……23
3.3.2 冷凍細胞……………………………………………………23
3.3.3 解凍細胞……………………………………………………23
3.3.4 細胞飢餓與病毒處理………………………… ……………24
3.4 蛋白質水解 (protein digestion) …………………………… ……24
3.4.1 蛋白質濃度定量 (protein assay) …………… ……………24
3.4.3 TCA 沉澱………………………………………………………24
3.4.4 穩定同位素標定……………………………………………25
3.4.5 C18 去鹽……………………………………………………25
3.4.6 強陽離子交換樹脂………………………………………………25
3.5 病毒試驗…………………………………………………………25
3.5.1 病毒增值………………………………………………………….25
3.5.2 病毒力價測定……………………………………………………26
3.6 西方墨點法……………………………………………………………26
3.6.1 蛋白質萃取………………………………………………………26
3.6.2 SDS-PAGE 製作………………………………………………27
3.6.3 SDS-PAGE 電泳、轉漬及blocking………………………………27
3.6.4 冷光呈色…………………………………………………………28
3.7 蛋白質身分鑑定與定量……………………………………………28

第四章 結果…………………………………………………………30-44
第五章 討論…………………………………………………………45-48
第六章 參考文獻……………………………………………………50-60
附錄……………………………………………………………………60-115
作者簡介

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