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研究生:官柏志
研究生(外文):BorJyh Guan
論文名稱:傳染性胰臟壞死病毒感染大眼鮭魚胚胎細胞株引發轉錄因子NF-kB之活化以造成細胞凋亡
論文名稱(外文):IPNV-Induced Apoptosis in CHSE-214 Cell Line via Activation of Transcription Factor NF-kB
指導教授:吳金洌吳金洌引用關係
指導教授(外文):JenLeih Wu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:漁業科學研究所
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:114
中文關鍵詞:傳染性胰臟壞死病毒細胞壞死細胞凋亡轉錄因子NF-kB大眼鮭魚胚胎細胞株Proteasome Inhibitor
外文關鍵詞:IPNVNecrosisApoptosisTranscription FactorNF-kBCHSE-214Proteasome Inhibitor
相關次數:
  • 被引用被引用:3
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  • 下載下載:19
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摘要
傳染性胰臟壞死病毒 (Infectious Pancreatic Necrosis Virus, IPNV)屬於兩段雙股核醣核酸病毒科,為一感染多種經濟性魚貝類且分佈廣泛之重要水生生物病毒。根據本實驗室先前之研究,證實IPNV感染大眼鮭魚胚胎細胞株 (CHSE-214) 後會引發細胞凋亡。為瞭解病毒感染造成宿主細胞凋亡之調控機制,本論文欲探討轉錄因子在IPNV引發細胞凋亡所扮演之角色,尤其是已知在其他系統的細胞凋亡扮演關鍵角色之轉錄因子nuclear factor-kappa B (NF-kB)與cyclic-AMP-response-element-binding protein (CREB),是否亦參與IPNV感染所引發之細胞凋亡。
利用electrophoretic mobility shift assay之分析,證實轉錄因子 NF-kB 與CREB 在病毒感染後8小時皆有明顯之活化現象;而tyrosine kinase抑制劑 (Genistein) 的添加可抑制此二轉錄因子之活化,證實tyrosine kinases在病毒感染所造成的細胞凋亡中扮演著關鍵的訊息傳遞角色。另利用Trans-activation assay之分析,佐證NF-kB的活化可促使含NF-kB結合序列的報告基因之表現,證實NF-kB之活化確可促進其下游基因之表現。進一步利用NF-kB活化抑制劑Proteasome inhibitor I或II之處理,繼之以IPNV感染8小時並以Hoechst 33258與Acridine Orange染劑觀察及計數細胞凋亡比例,顯示其可從僅以IPNV感染之43.1 %,降至抑制劑I或II處理後之12.5 % 及14.7 % ,證實NF-kB之活化可促進細胞之凋亡。而在西方點墨法之分析結果發現,NF-kB之活化對病毒蛋白的表現並未有促進或抑制之效果。
綜合上述結果,證實IPNV感染大眼鮭魚胚胎細胞株 (CHSE-214)所造成的細胞凋亡,必須經由tyrosine kinases傳遞訊息,並進一步活化轉錄因子NF-kB而促使細胞凋亡。
Abstract
Infectious Pancreatic Necrosis Virus (IPNV) belonging to the Birnaviridae with bisegmented double-strand RNA genomes is one of the widespread fish pathogen and could infect many economically important finfish and shellfish. According to previous study in our laboratory, IPNV infection could induce apoptosis in chinook salmon embryo cell line CHSE-214. To clarify the regulation mechanism in IPNV-induced apoptosis, this thesis is attempted to identify host cell transcription factors involved in this apoptosis response, especially nuclear factor-kappa B (NF-kB) and cyclic-AMP-response-element-binding protein (CREB) that are well known to play important roles in the regulation of apoptosis in other systems.
As determined in electrophoretic mobility shift assays, IPNV infection in CHSE-214 cells leads to activation of NF-kB and CREB at 8 hours post infection. Treatment of IPNV-infected CHSE cells with a tyrosine kinase inhibitor (Genistein) prevents NF-kB and CREB activation and suggests tyrosine kinases are key regulators in signaling pathway of IPNV-induced apoptosis. IPNV-induced activation of NF-kB DNA-binding activity correlated with the onset of NF-kB-directed expression of reporter gene in trans-activation assay. Treatments of IPNV-infected CHSE cells with proteasome inhibitor I and II inhibit NF-kB activation and substantially diminish IPNV-induced apoptosis from 43.1 % to 12.5 % and 14.7 % which were quantitated by Hoechst 33258 and Acridine Orange staining. Finally, these two inhibitors have no influence on viral protein expression of IPNV by Western Blotting.
These findings indicate that the signaling of IPNV-induced apoptosis in CHSE-214 cells is transmitted by tyrosine kinases and via the activation of transcription factor NF-kB.
中文摘要 1
英文摘要 3
壹、前言 4
貳、緒論 5
( 一 ) 傳染性胰臟壞死病毒之基本特性 5
( 1 ) 傳染性胰臟壞死病毒之發現史與分類
( 2 ) 傳染性胰臟壞死病毒宿主種類與臨床症狀
( 3 ) 傳染性胰臟壞死病毒之血清型分類與分類情形
( 4 ) 傳染性胰臟壞死病毒之生長與生化特性
( 5 ) 傳染性胰臟壞死病毒遺傳物質之研究
( 6 ) 傳染性胰臟壞死病毒的蛋白質及其功能
( 7 ) 傳染性胰臟壞死病毒之複製與轉錄
( 8 ) 傳染性胰臟壞死病毒之轉譯
( 9 ) 台灣地區傳染性胰臟壞死病毒之相關研究
( 二 ) 病毒引發細胞凋亡之研究 18
( 1 )細胞壞死與細胞凋亡
( 2 )細胞凋亡的生理意義
( 3 )細胞凋亡的訊息傳遞與調控
( 4 )病毒感染與細胞凋亡
( 5 )IPNV引發魚類細胞株之細胞凋亡研究
( 三 ) 轉錄因子NF-kB與細胞凋亡之研究 25
( 1 )NF-kB/Rel蛋白家族的發現與結構特性
( 2 )NF-kB的活化與其功能的調節
( 3 )NF-kB在調控細胞凋亡上所扮演的角色
參、實驗材料 32
一、材料與試藥
( 一 ) 生物材料 32
( 二 ) 細胞及病毒培養液 32
( 三 ) 生物反應試劑組 33
( 四 ) 反應溶液及緩衝溶液 33
二、儀器和器材 37
肆、實驗方法 38
一、細胞培養與病毒增殖 38
二、NF-kB及CREB 的Gel shift assay
三、NF-kB trans-activation assay
四、利用Hoechst 33258與Acridine Orange染劑染色
判定凋亡細胞
五、西方點墨法
伍、實驗結果 48
( 一 ) IPNV感染魚類細胞株引發轉錄因子NF-kB的活化 48
( 二 ) IPNV感染魚類細胞株引發轉錄因子CREB的活化 49
( 三 ) Tyrosine kinase抑制劑Genistein能抑制
NF-kB與CREB活化 50
( 四 ) IPNV感染魚類細胞後NF-kB transactivation的變化 51
( 五 ) NF-kB之活化扮演著促進宿主細胞凋亡的功能 53
( 六 ) NF-kB之活化不會影響病毒蛋白之表現 59
陸、討論 61
柒、結論 71
捌、參考文獻 72
玖、圖表 90
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