臺灣博碩士論文加值系統

本省海水魚類之虹彩病毒感染症，經常爆發在春、夏季水溫較高時，因此，本論文擬由三方面來探討溫度因子對台灣石斑虹彩病毒 (以下簡稱TGIV) 致病機制之影響。
首先進行in vivo感染實驗，以釐清溫度因子對TGIV毒力之影響。將石斑魚苗以TGIV (104 TCID50/fish) 進行腹腔注射後，分別飼養於18、24、以及30℃，結果發現，飼養在24和30℃下的感染魚，兩週後的累計死亡率分別達93% 和70%，而18℃下感染魚的累計死亡率只有20%；此外由各組的死亡曲線，發現18℃的低水溫，顯然能延緩病毒感染造成魚苗死亡的發生。此外，進一步比較不同溫度下增殖之TGIV的致病力，分別收集培養在18、24、與30℃的病毒，並以腹腔注射方式感染石斑魚苗，結果兩週後三組病毒所造成的死亡率皆達100%，顯示此三個培養溫度下所增殖的TGIV對石斑魚苗皆有相當高的致病力。其次以光顯染色觀察TGIV在18、24、和30℃下所引發SB細胞株之細胞病變情形，結果三個培養溫度的細胞變化有所分歧。病毒接種四天後，30℃下的細胞多數已出現嗜鹼性包含體、細胞變圓拱起、空泡化、細胞萎縮以及死亡等現象；在24℃時則可見大多數細胞內有嗜鹼性包含體，而死細胞數目明顯較少；至於18℃時細胞病變情況不明顯，且到八天後仍有正常細胞存在。再者，測定以上三個培養溫度中TGIV的增殖曲線，發現TGIV在不同培養溫度下，細胞內的增殖曲線相仿，細胞相關病毒的力價最終皆可達108 TCID50/ml；但30℃時病毒的釋放較其他兩組快，第五天釋放病毒力價為106 TCID50/ml，高出另外兩組約100倍，顯示此三種培養溫度雖對TGIV病毒的增殖無明顯影響，但30℃則有助於病毒釋放。
另一方面，為研究溫度與石斑虹彩病毒對石斑魚非特異性免疫系統之影響，取石斑魚頭腎細胞培養在18、24、與30℃，並利用NBT法測定細胞呼吸爆活性。結果顯示，石斑魚頭腎細胞的呼吸爆活性對溫度敏感，在較高的溫度下 (24、30℃) 只有短期間仍可維持活性，24小時後呼吸爆活性顯著下降；另一方面，雖然在較低溫的18℃溫度下，頭腎細胞的呼吸爆活性能維持48小時，但是在TGIV感染後頭腎細胞產生呼吸爆的能力則明顯降低。
由於TGIV 於18、24、以及30℃下引起SB細胞的變化情形不同，為了進一步釐清溫度和TGIV是否會誘發細胞凋亡，分別就SB和頭腎細胞，進行DAPI染色和DNA的梯狀片段分析。DAPI染色之觀察結果顯示，30℃溫度下，TGIV感染後確實會導致SB細胞核出現細胞凋亡特徵，有別於18℃和24℃下細胞之變化情形。30℃下病毒接種3天後，細胞出現核內染色質邊緣化和染色質濃縮等變化，第5天此種現象更為明顯；同樣地，30℃下感染細胞的DNA也呈現梯狀片段化的情形。但是培養18℃和24℃的病毒感染細胞，和以上三個溫度的控制組皆無此現象。另外以石斑魚頭腎細胞進行DAPI染色和DNA梯狀片段化分析，結果三個培養溫度的細胞無論是否感染TGIV，皆有細胞具細胞凋亡特徵，而各組間並無差別。
綜合以上結果，推測高水溫 (30℃) 雖然對病毒的增殖沒有影響，但在高溫有利於TGIV釋放，而此釋放之機制可能與病毒引發細胞凋亡有關。

中文摘要..................................................Ⅰ
英文摘要..................................................Ⅲ
目錄......................................................Ⅵ
前言.......................................................1
文獻整理...................................................4
一、新興魚類虹彩病毒感染症之簡介..........................4
二、溫度與魚體免疫能力、病毒致病機制之關係................6
(一) 溫度對魚體免疫能力的影響.............................6
(二) 溫度對魚類病毒病原性之影響..........................10
三、細胞凋亡之簡介.......................................13
(一) 生理意義............................................13
(二) 魚類非特異性免疫能力與細胞凋亡的關係................14
(三) 細胞凋亡在病毒與寄主間的角色........................15
材料與方法................................................18
一、溫度因子對石斑虹彩病毒毒力之影響.....................18
(一) 實驗材料............................................18
(二) 研究方法............................................21
實驗一、溫度因子對TGIV致病力之影響.......................21
1、TGIV感染魚在不同飼養溫度之死亡情形...................21
2、不同溫度下增殖之TGIV之致病力.........................21
實驗二、溫度因子對TGIV感染力之影響.......................22
1、以光顯染色觀察在TGIV不同溫度所引發之細胞病變情形.....22
2、TGIV 在不同培養溫度之增殖情形........................22
二、溫度與石斑虹彩病毒對石斑魚頭腎細胞吞噬活性之影響.....23
(一) 實驗材料............................................23
(二) 研究方法............................................23
1、石斑魚頭腎細胞之分離.................................23
2、96孔微量平板製備與接種病毒...........................24
3、頭腎細胞呼吸暴活性分析－NBT反應......................24
三、溫度與石斑虹彩病毒誘發細胞凋亡之觀察.................24
(一) 實驗材料............................................25
(二) 研究方法............................................25
1、DAPI螢光染色.........................................25
2、細胞DNA梯狀片段分析..................................26
結果......................................................27
一、溫度因子對石斑虹彩病毒毒力之影響.....................27
(一)、溫度因子對TGIV致病力之影響.........................27
1、TGIV感染魚在不同飼養溫度之死亡情形...................27
2、不同溫度下增殖之TGIV之致病力.........................27
(二)、溫度因子對TGIV感染力之影響.........................28
1、以光顯染色觀察TGIV在不同溫度所引發之細胞病變情形.....28
2、TGIV 在不同培養溫度之增殖情形........................29
二、溫度與石斑虹彩病毒對石斑魚頭腎細胞吞噬活性之影響.....29
三、溫度與石斑虹彩病毒誘發細胞凋亡之觀察.................30
(一)、利用DAPI螢光染螢光染色觀察細胞核型態之變化.........30
1、SB細胞之觀察結果.....................................30
2、石斑魚頭腎細胞之觀察結果.............................31
(二)、萃取細胞DNA進行梯狀片段分析........................31
1、SB細胞...............................................31
2、石斑魚頭腎細胞.......................................31
討論......................................................32
參考文獻..................................................38
圖........................................................49

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