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研究生:周宜黎
研究生(外文):Yi-Li Chou
論文名稱:傳染性胰臟壞死病毒凋亡基因vp3所誘發細胞凋亡分子機制之研究
論文名稱(外文):Molecular apoptotic mechanism of infectious pancreatic necrosis virus vp3
指導教授:洪健睿洪健睿引用關係
指導教授(外文):Jiann-Ruey Hong
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
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:2004
畢業學年度:92
語文別:中文
論文頁數:78
中文關鍵詞:細胞凋亡傳染性胰臟壞死病毒
外文關鍵詞:IPNVapoptosis
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  根據本實驗室之研究,證實傳染性胰臟壞死病毒感染大眼鮭魚胚胎細胞株後會誘發細胞凋亡。但是病毒本身之基因是否參與誘發宿主細胞凋亡尚不完全清楚。於初步測試中,發現大量表現VP3會導致細胞快速凋亡。因此本論文就計劃針對VP3基因功能作進一步探討,想瞭解傳染性胰臟壞死病毒中之VP3基因是如何誘發細胞凋亡及可能之分子機制。
  首先利用西方點墨法,發現在3T3細胞株內額外大量表現VP3至24小時中,發現可以誘導Bad基因表現,但對Bcl-2和Bax等基因則沒有明顯的影響。接著針對caspase-8和-3之受質來測試caspase-8和-3的活化情形。結果caspase-8在12小時明顯的大量被活化,同時caspase-3也在第24小時逐漸被活化,於72小時達到最高峰。接著利用專一性之caspase的抑制劑來抑制caspase-8及-3之活性,結果發現caspase-8及-3之專一性抑制劑在12和24小時有良好之抑制效果,並能阻段具有活性caspase-8與-3之形成和延遲細胞凋亡的現象產生。
  綜合以上的結果顯示,大量表現VP3能誘發3T3細胞株內的Bad蛋白質大量表現,及活化細胞內的caspase-8和下游的caspase-3,進而使得細胞走向凋亡。我們希望藉由此研究能進一步瞭解傳染性胰臟壞死病毒誘發細胞凋亡之分子機制,能有助於對傳染性胰臟壞死病毒所造成之疾病的預防和治療。
  From the previous study, the Infectious Pancreatic
Necrosis Virus (IPNV) could induces Chinook salmon embryo cell (CHSE-214) death through apoptosis, but whether viral gene could involved in host cell death still don’t known. On the other hand, from our preliminary data shown that we found overexpression the vp3 could rapidly induce 3T3 cell death after 24hr incubation. So in this study,we want to examine vp3 how to induce apoptotic death and its molecular-death mechanism in 3T3 cells.
  Interestingly,we found that the bad gene could be induced at 24hr post-incubation time,but did not in Bcl-2 or Bax genes during vp3 overexpression in 3T3 cells. Then, initiator of caspase-8 and effector of caspase-3 also could be activated at either 12hr or 24hr post-incubation time, respectively. Finally, we found that specific caspase-8 and -3 inhibitors all could prevent the cell death and block the active forms of caspase-8 and -3 formation from precursor forms.
  In summary,the vp3 gene induces the 3T3 apoptotic cell death that may be mediated the pro-apoptotic gene bad expression to activate the caspase-8 and -3 for triggered the cell death,which could be useful for rational design of strategies for the prevention and /or treatment of IPNV induces diseases.
致謝…………………………………………………………………… Ⅰ
中文摘要……………………………………………………………… Ⅱ
英文摘要……………………………………………………………… Ⅲ
目錄…………………………………………………………………… Ⅳ
表圖目錄……………………………………………………………… Ⅵ
第一章、緒論………………………………………………………… 1
第一節、傳染性胰臟壞死病毒之基本特性…………………………1
第二節、病毒誘發宿主細胞凋亡(apoptosis)之研究………… 8
第三節、Bcl-2蛋白質家族與調控細胞凋亡之研究………………13
第四節、Caspase與誘發細胞凋亡之研究…………………………17
第二章、材料與方法………………………………………………… 20
第一節、材料…………………………………………………………20
第二節、儀器…………………………………………………………23
第三節、方法……………………………………………………… 25
第三章、結果……………………………………………………………31
第一節、vp3與EGFP基因轉染3T3細胞株……………………… 31
第二節、vp3基因誘發3T3細胞株的凋亡………………………… 31
第三節、vp3基因轉染3T3細胞株所造成Bcl-2家族蛋白質的變化 33
第四節、vp3基因轉染3T3細胞株所造成Caspase家族
蛋白質的變化…………………………………………………35
第四章、討論……………………………………………………………38
第一節、魚類細胞株基因轉染效率低………………………………38
第二節、專一性抗體於魚類細胞株辨識能力低……………………39
第三節、vp3表現對誘導Bcl-2家族之研究…………………………39
第四節、vp3表現活化Caspases之研究…………………………… 40
第五節、未來之展望…………………………………………………41
參考文獻…………………………………………………………………43
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