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研究生:劉興懿
研究生(外文):Hsin-I Liu
論文名稱:台灣石斑虹彩病毒誘導石斑魚泳鰾細胞發生溫度相關凋亡之研究
論文名稱(外文):Occurrence of temperature-dependent apoptosis of grouper swimming bladder cells induced by grouper iridovirus of Taiwan (TGIV)
指導教授:周信佑周信佑引用關係
指導教授(外文):Hsin-Yiu Chou
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
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:2007
畢業學年度:95
語文別:中文
論文頁數:62
中文關鍵詞:虹彩病毒石斑魚細胞凋亡溫度
外文關鍵詞:IridovirusGrouperApoptosisTemperature
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摘 要

本論文針對台灣石斑虹彩病毒 (Grouper iridovirus of Taiwan, TGIV),探討溫度對其致病之影響以及細胞凋亡在其中的角色。首先將石斑魚苗以TGIV (104 TCID50/fish)進行腹腔注射後,分別飼養於18℃、25℃、32℃三個溫度中,兩週後的累積死亡率分別為45%、100%及78%,而對照組在三個溫度下皆沒有死亡。由於32℃死亡率反而下降,因此本論文亦探討了TGIV在不同溫度下誘發細胞凋亡的情形,以釐清死亡率下降之因素。在32℃下病毒感染組出現細胞核濃縮等細胞凋亡現象。同樣32℃病毒感染的細胞,在超微上可見有疑似凋亡小體脂囊泡、細胞核內染色質濃縮成塊的情形。TUNEL染色結果也發現,32℃下TGIV感染SB細胞於感染後第一天便開始出現陽性反應,到第七天時有47.62%的細胞出現陽性反應;而18℃及25℃之感染組,陽性反應分別只佔了0.58%與1.02%。以Caspase抑制劑處理感染細胞,發現32℃下凋亡細胞數量明顯降低,但仍有細胞病變效應的出現。另外32℃感染組細胞於第三天,出現微量的DNA梯狀片段化圖形,同時隨著時間增長,片段化情形更加明顯;而18℃及25℃感染組,則沒有明顯片段化情形。利用PI染色經流式細胞儀分析細胞週期,32℃高溫感染組於感染後5天,出現細胞凋亡的Sub G1時期,在第六天時則有明顯的peak出現。最後進行in vivo實驗,將三個溫度下TGIV感染瀕死魚隻的心、頭腎、肝、脾及鰓,分別進行DNA梯狀片段化分析,結果32℃感染組的各臟器都出現了梯狀片段化的情形,而18℃以及25℃之感染組以及控制組,則沒有明顯片段化的情形。以上in vitro和in vivo結果顯示,TGIV會誘發細胞凋亡的發生,而此現象與溫度有密切關連,推測可能為溫度相關凋亡 (temperature-dependent apoptosis)。
Abstract

Aiming at Grouper Iridovirus of Taiwan (TGIV), this research discusses the correlation among temperature, viral pathogenicity and virus-induced apoptosis. First, grouper were intraperitoneal injected with TGIV in concentrations of 103.5 TCID50/fish, and then reared at 18℃, 25℃ and 32℃ respectively. After 2 weeks, the cumulative mortality of virus-infected fish under the above 3 water temperature were 45%, 100%, 78% respectively and control fish were not found death in these three temperatures. The cytopathic effect (CPE) of TGIV inoculated SB cell, revealed cell death at 32℃ was faster than at 25℃. It was suspected apoptosis at 32℃. Therefore TGIV induce apoptosis at different temperature were be analyzed. Under DAPI fluorescence staining, we found the fish of virus-infected appeared apoptosis like pyknosis. Further we use transmission electron microscopy to observe the virus-infected cell at 32℃ seems to have blebbing like apoptotic body and nuclear margination. It is also found in TUNEL staining, TGIV-infected SB cell of 32℃ showed lighter positive signal at the first day post-infected (dpi), and the numbers of positive cell reach 47.62% until 7dpi. Otherwise the positive signal in the virus-infected SB cell at 18℃ and 25℃ account for 0.58% and 1.02% respectively. The numbers of apoptotic cell at 32℃ decrease apparently when treated with caspase inhibitor handle with infected cell. Besides, 32℃’s infected cell showed pattern from 3dpi and result of 7dpi was more apparent. There are obvious DNA laddering of virus-infected cell at 18℃ and 25℃. PI stained cell were analysis with flow cytometry, the virus-infected cell at 32℃ show up apoptosis in sub G1 phase at 5dpi. The apoptotic cell number increase at 6dpi. DNA laddering was also carried out in vivo experiment. Organs (heart, head kidney, spleen, and gill) of TGIV-injected fish reared at 32℃ show DNA laddering pattern as well. And DNA laddering was not found in fish reared 18℃, 25℃ and control group. According to the result obtained in vitro and in vivo, TGIV-induced apoptosis was found closed related with temperature, figuring out it might be temperature-dependent apoptosis.
目 錄
中文摘要 i
英文摘要 iii
謝辭 v
目錄 vi
圖目錄 viii
表目錄 ix
前言 1
文獻整理 3
一、魚類虹彩病毒感染症之簡介 3
二、水產動物病毒之致病機制與細胞凋亡 6
三、細胞凋亡之簡介與分析方法 9
四、細胞凋亡之途徑 13
五、溫度效應與魚類病毒的致病力 16
材料與方法 19
一、實驗材料 19
(一) 細胞株 19
(二) 台灣石斑虹彩病毒 21
(三) 實驗用魚 22
二、研究方法 22
(一) 溫度對石斑虹彩病毒致病力之影響 22
(二) 三種溫度下石斑虹彩病毒誘發SB細胞凋亡之觀察 22
1、DAPI螢光染色 22
2、電子顯微鏡術觀察 23
3、TUNEL測定法 24
4、細胞DNA梯狀片段化分析 25
5、以流式細胞儀測定細胞週期及細胞凋亡 25
6、Caspase抑制劑處理TGIV感染之SB細胞株 26
7、臟器DNA梯狀片段化分析 26
結果 28
一、溫度因子對石斑虹彩病毒致病力之影響 28
二、三種溫度下石斑虹彩病毒誘發SB細胞凋亡之觀察 28
(一)、DAPI螢光染色觀察細胞核形態之變化 28
(二)、電子顯微鏡術觀察 28
(三)、TUNEL測定法 29
(四)、DNA梯狀片段化分析 29
(五)、以流式細胞儀測定細胞週期及細胞凋亡 30
(六)、Caspase抑制劑對TGIV所誘發細胞凋亡之觀察 30
三、感染魚之臟器DNA梯狀片段化分析 31
討論 32
參考文獻 38
圖 50
表 63
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