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研究生:林瑩祝
研究生(外文):Ying-Chu Lin
論文名稱:高溫誘發台灣石斑虹彩病毒在invitro與invivo之細胞凋亡研究
論文名稱(外文):Studies of Grouper iridovirus of Taiwan (TGIV) induce apoptosis in vitro and in vivo
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:2009
畢業學年度:97
語文別:中文
論文頁數:97
中文關鍵詞:臺灣石斑虹彩病毒細胞凋亡高溫石斑魚
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首先觀察18、25、32℃三種溫度下對台灣石斑虹彩病毒 (Grouper iridovirus of Taiwan, TGIV) 增殖的影響,初步以PCR偵測病毒的增殖情形,結果發現18℃、25℃下可以明顯偵測到TGIV,而32℃下則幾乎偵測不到病毒的存在;之後分析以上三個溫度下TGIV之增殖曲線,結果18℃、25℃的細胞相關病毒 (Cell associate virus, CAV) 在感染後16天力價達106∼107 TCID50/ml,32℃只有104.25 TCID50/ml;另外釋放性病毒 (Release virus, RV) 力價以25℃達106.4 TCID50/ml最高,18℃與32℃只有104 TCID50/ml左右。之後以PI與Annexin-V染色經流式細胞儀分析細胞週期與凋亡細胞,發現在32℃高溫感染組於感染後五天,開始出現細胞凋亡的Sub G1時期,第九天時細胞週期中Sub G1波峰更為明顯,數量化後凋亡細胞比例約佔37.5%;而Annexin-V與PI雙重染色則可發現25℃感染組的細胞,隨著感染天數增加逐漸往四象限圖中壞死細胞代表的Q1+Q2位置移動,32℃則逐漸往凋亡細胞代表的Q4象限移動,感染後第九天凋亡比例達24%。另外Annexin-V螢光染色觀察也可以發現18℃與25℃於感染後第三天開始出現PI染色發出紅色螢光的壞死細胞,而32℃感染後第三天則是出現Annexin-V染色發出綠色螢光的凋亡細胞。進一步以西方點墨法 (Western blot) 分析高溫下TGIV感染SB細胞之熱休克蛋白70、抗凋亡蛋白Bcl-2與促凋亡蛋白Bak的表現,結果32℃感染組熱休克蛋白70有明顯表現量,但是在抗凋亡蛋白Bcl-2與促凋亡蛋白Bak的表現都不明顯;相對地,25℃感染組則在抗凋亡Bcl-2蛋白大量表現,但熱休克蛋白70與促凋亡蛋白Bak的表現不明顯。在活體實驗中,32℃病毒攻擊組兩週後的累積死亡率只有37.5%,25℃累積死亡率卻達到100%,將死亡魚隻進行光學組織切片H&E染色與TUNEL染色後觀察,同樣在32℃組呈現較強的細胞凋亡TUNEL陽性反應,顯示高溫下TGIV誘發之細胞凋亡可能與細胞防禦病毒感染機制相關。
The influence of temperature on the Grouper iridovirus of Taiwan (TGIV) multiplication at 18, 25 and 32℃, respectively were investigated in the study. First, PCR results showed TGIV-infected cells at 18 and 25℃ have 1339 bp specific TGIV amplicon but could not be found at 32℃. Then cell-associated virus (CAV) and released virus (RV) at different time points after TGIV inoculation was estimated for determining the growth curve of TGIV. Virus yields of CAV under hyperthermia (32℃) group only 104.25 TCID50/ml, lower than the other two temperature groups of titer 106∼107 TCID50/ml. Furthermore, cell cycles and apoptosis cells were measured by flow cytometric analysis using propidium iodide (PI) and Annexin-Ⅴ. The TGIV-infected cells at 32℃ showed apoptosis in the sub-G1 phase at 5 dpi and reached peak at 9 dpi. And it had 24% apoptosis cells at 9 dpi in Q4 region. The double labeling assay method combines the staining properties of Annexin-Ⅴ and PI was observed by microscopy. The test detected apoptosis-dependent phosphatidylserine translocation from the inner to the outer leaflet of the plasma membrane at 32℃. Western blot assays analyzed heat-shock protein 70 (HSP 70), anti-apoptosis protein bcl-2 and pro-apoptosis protein bak expression of TGIV-infected cells at the above three temperatures. There are obvious expressions of HSP 70 and pro-apoptosis protein bak at 32℃. And anti-apoptosis protein bcl-2 was found expressing significantly at 25℃ only. Finally, fish were injected TGIV and held at 25 and 32℃. After 2 weeks, the cumulative mortality of virus-infected fish was 100% and 37.5% respectively. Besides, the 32℃ group showed more obvious TUNEL positive reaction than 25℃ group. The results revealed that hyperthermia-induced apoptsis might closely related with host defense mechanism against TGIV infection.
謝辭 i
中文摘要 ii
英文摘要 iv
目錄 vi
圖目錄 viii
前言 1
文獻整理 3
一、魚類虹彩病毒感染症之簡介 3
二、溫度與水產動物病毒致病之關連 6
三、水產動物病毒與細胞凋亡 10
四、細胞凋亡之簡介與分析法 13
五、細胞凋亡與熱休克蛋白 20
材料與方法 23
一、實驗材料 23
(一) 細胞株 23
(二) 台灣石斑虹彩病毒 24
(三) 實驗用魚 25
(四) 感染實驗用之循環控溫養殖系統 25
二、研究方法 26
(一) 不同溫度對石斑虹彩病毒增殖力影響 26
1、PCR測定 26
2、RV之收集 27
3、CAV之收集 27
(二) 不同溫度下石斑虹彩病毒在SB細胞株中凋亡之觀察 27
1、以流式細胞儀分析細胞週期及凋亡 27
(1) PI染色 27
(2) Annexin-Ⅴ染色 28
2、Annexin-Ⅴ螢光染色 28
3、Western blot偵測熱休克蛋白70、Bcl-2與Bak蛋白表現 28
(三) 不同溫度下臺灣石斑虹彩病毒感染對魚體中組織所誘發凋亡情形 30
1、虹彩病毒感染石斑魚之組織病理觀察 31
2、虹彩病毒誘發石斑魚細胞凋亡分析 32
結果 33
一、不同溫度對石斑虹彩病毒增殖力影響 33
(一) PCR測定 33
(二) 病毒增殖曲線圖 33
二、不同溫度下石斑虹彩病毒在SB細胞株中凋亡之觀察 34
(一)、以流式細胞儀分析細胞週期及凋亡細胞 34
1. PI染色觀察細胞週期變化 34
2. Annexin-Ⅴ與PI染色觀察凋亡細胞產生之情形 34
(二)、Annexin-Ⅴ與PI螢光染色觀察凋亡細胞與壞死細胞 35
(三)、Western blot偵測熱休克蛋白70、Bcl-2與Bak蛋白表現 36
1. 熱休克蛋白70表現情形 36
2. Bcl-2蛋白質表現情形 36
3. Bak蛋白質表現情形 36
三、不同溫度下臺灣石斑虹彩病毒於魚體中組織所誘發凋亡情形 37
(一)、利用H & E染色觀察虹彩病毒感染石斑魚之組織病理切片 37
(二)、利用TUNEL染色分析虹彩病毒誘發石斑魚細胞凋亡反應 37
討論 39
參考文獻 44
圖 62
表 89

圖 目 錄

圖一、不同溫度下病毒增殖PCR結果 62
圖二、各種不同溫度下病毒增殖曲線測定 63
圖三、以流式細胞儀分析18℃感染細胞之細胞週期 64
圖四、以流式細胞儀分析25℃感染細胞之細胞週期 65
圖五、以流式細胞儀分析32℃感染細胞之細胞週期 66
圖六、以流式細胞儀分析細胞週期之量化結果 67
圖七、以流式細胞儀分析18℃感染細胞之凋亡情形 68
圖八、以流式細胞儀分析25℃感染細胞之凋亡情形 69
圖九、以流式細胞儀分析32℃感染細胞之凋亡情形 70
圖十、以流式細胞儀分析凋亡細胞之量化結果 71
圖十一、以流式細胞儀分析壞死細胞之量化結果 72
圖十二、以螢光顯微鏡觀察凋亡與壞死細胞產生之情形 73
圖十三、以Western blot偵測25℃與32℃熱休克蛋白70表現情形 74
圖十四、以Western blot偵測25℃與32℃抗凋亡蛋白Bcl-2表現情形 75
圖十五、以Western blot偵測25℃與32℃促凋亡蛋白Bak表現情形 76
圖十六、溫度因子對病毒致病力影響 77
圖十七、25℃下罹病魚之組織病理切片H & E染色全圖 78
圖十八、25℃下罹病魚之組織病理切片各臟器H & E染色圖 79
圖十九、25℃下罹病魚之組織病理切片凋亡反應TUNEL染色全圖 80
圖二十、25℃下罹病魚之組織病理切片各臟器凋亡反應TUNEL染色圖 81
圖二十一、32℃下罹病魚之組織病理切片H & E染色全圖 82
圖二十二、32℃下罹病魚之組織病理切片各臟器H & E染色圖 83
圖二十三、32℃下罹病魚之組織病理切片凋亡反應TUNEL染色全圖 84
圖二十四、32℃下罹病魚之組織病理切片各臟器凋亡反應TUNEL染色圖 85
圖二十五、不同溫度各臟器細胞凋亡TUNEL陽性反應數量統計表 86
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