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研究生:洪健睿
研究生(外文):Jiann-Ruey Hong
論文名稱:感染性胰臟壞死病毒所誘發寄主細胞凋亡及分子機制之研究
論文名稱(外文):Induction and molecular regulation of host cell apoptosis by infectious pancreatic necrosis virus infection
指導教授:吳金洌吳金洌引用關係
指導教授(外文):Jen-Leih Wu
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:英文
中文關鍵詞:感染性胰臟壞死病毒細胞自戕鮭魚細胞株VP5蛋白病毒複製後潰瘍壞死電子顯微鏡Bcl-2家族
外文關鍵詞:Infectious pancreatic necrosis virusApoptosisCHSE-214 cellsVP5 proteinvirus replicationsecondary necrosiselectron microscopeBcl-2 family
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中文摘要
細胞凋亡是一種主動過程化的細胞死亡方式,並在高等生物裡於特定生理條件下扮演多種功能,其發生過程受到嚴謹的控制著。生物體常為了生存而以細胞凋亡方式來清除有害細胞,例如在機制上利用細胞凋亡方式將受到病毒感染的細胞消除。
感染性胰臟壞死病毒是一種能感染具有經濟性重要魚類的病毒,它是屬於水生二段式核醣核酸病毒科的病毒。E1-S病毒是從日本病鰻在台灣分離出來,歸屬於胰臟壞死病毒Ab血清型的病毒株。
於本論文中,主要利用鮭魚細胞株和E1-S病毒為材料,針對E1-S與細胞宿主凋亡之現象,提出幾個問題並嚐試回答該問題。問題一:E1-S是否能誘發鮭魚宿主細胞凋亡。首先我們是第一個在世界上發現胰臟壞死病毒能誘發鮭魚宿主細胞凋亡的例子,證實的方式主要利用電子顯微鏡的技術分析被E1-S感染的細胞株。主要從不同時段的細胞型態改變來判斷,如細胞出現高度聚合的染色質,脆裂的細胞核和染色質邊緣化的現象,並於最後階段中發現從前潰瘍性細胞中出現第二階段的潰瘍性死亡。問題二:E1-S病毒感染後的宿主凋亡是否需要透過病毒基因複製才能進行。基本上引發宿主凋亡是需要病毒基因的複製才能引發的,且利用特定藥劑之添加於抑制病毒基因之複製,即能阻斷E1-S病毒感染後的宿主凋亡。問題三:病毒VP5蛋白於病毒感染後早期階段是否具有抗宿主凋亡之現象。從VP5氨基酸的序列與Bcl-2的家庭成員比對時,於重要的四個區段裡發現有高度相似性,其應屬於新的Bcl-2家庭成員之一,同時其扮演具有調節病毒表現的能力和病毒感染早期抗宿主凋亡的雙功能角色。
從本論文的研究中,希望能在感染性胰臟壞死病毒的致病機制上能提供一些新的見識,以增進對該病毒之瞭解。希望能在未來有利於感染性胰臟壞死病毒之預防和治療。
English abstract
Apoptosis is an active process of cell death that serves diverse functions in multicellular organisms, and under physiological conditions, it is tightly controlled. Apoptosis is often implicated in the elimination of cells whose survival might be harmful to organism as a whole; for example, the mechanisms have evolved to target virus-infected cells for apoptotic cell death. Infectious pancreatic necrosis virus (IPNV) is an economically important fish pathogen and belongs to Birnaviridae virus family. E1-S of IPNV Ab strain was isolated from Japanese eel in Taiwan. In this Ph. D. Thesis, we asked several questions and examined their possibilities that (1) whether the E1-S can induce apoptotic cell death of host cells, and (2) whether the E1-S virus replication is required for triggering host cell death. Finally, we asked what is the possible mechanism that the VP5 protein is able to delay the anti-host apoptotic cell death during IPNV infection. First, we found that the IPNV-infected CHSE-214 cells could induce apoptotic cell death of host cells. The apoptotic cells were analyzed with an electron microscope and showed a pattern of ultrastructural change, indicating that apoptosis appears before pathological changes of necrosis, including condensed chromatin, fragmented nuclei, nuclei with chromatin marginations, and secondary necrosis from prenecrotic cells. Second, the apoptotic cell death of host cells could be blocked when viral gene expression was inhibited by some potent drugs as cycloheximide, genistein and EDTA. Lastly, comparing the amino acids sequences with Bcl-2 family data showed that the VP5 is a new Bcl-2 family member, and plays a dual function on the regulation of viral replication and the defense of host suicide. Our studies may provide some insights into the pathogenesis of IPNV infection.
COVER
Acknowledgments
Contents
List of figures
Abbreviations
English abstract
Chapter 1.Introduction
1.1 Molecular biology of IPNV
1.1.1 Virus growth
1.1.2 Biophysical and biochemical characteristics
1.1.3 Steps on virus replication
1.2 Virus induce host apoptotic cell death
1.2.1 Morphological changes on apoptosis
1.2.2 Viruses that induce apoptosis
1.2.3 Viruses that block apoptosis
Chapter 2.Induction of apoptosis by infectious pancreatic necrosis virus in fish embryonic cells
Abstract
2.1 Induction of apoptosis and secondary necrosis by infectious pancreatic necrosis virus in fish embryonic cells
2.2 Apoptosis precedes necrosis of fish cell line with infectious pancreatic necrosis virus infection
2.3 Dynamics of nontypical apoptotic morphological changes visualized by green fluorescent protein in living cells with infectious pancreatic necrosis virus infection
Chapter 3.Molecular regulation of apoptosis by infectious pancreatic necrosis virus in fish embryonic cells
Abstract
3.1 Infectious pancreatic necrosis virus induces apoptosis due to down-regulation of sruvival factor Mcl-1 protein expression in fish cell line
3.2 Cloning and functional analysis of VP5:A novel Bcl-2 family with regulation of viral proteins expression and anti-apoptotic death activity
Chapter 4.Conclusion and discusion
4.1 Conclusion
4.2 Discusion:Future directions--from off-on system to antisense technology
4.2.1 On regualtion of host cell death by viral proteins
4.2.2 Anti-IPNV replication by the antisense technology in in Vitro and in vivo system
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