錦鯉疱疹病毒（Koi herpesvirus, KHV）目前被分類為水生動物疱疹病毒科（Alloherpesviridae）下的鯉科第三型疱疹病毒（Cyprinid herpesvirus 3, CyHV-3）。自1998年高致死率的錦鯉疱疹病毒傳播開後，嚴重打擊世界各地食用鯉魚以及錦鯉的繁養殖業，台灣也在2002年12月檢疫為錦鯉疱疹病毒疫區。由於目前台灣地區尚未建立適合的細胞可供錦鯉疱疹病毒分離使用，使得台灣的錦鯉疱疹病毒
研究停滯不前。本實驗建立了兩株錦鯉鰭細胞株，分別命名為錦鯉鰭細胞株1（KF1）及錦鯉鰭細胞株2（KF2）並且進行特性分析。兩株錦鯉鰭細胞株皆為表皮樣細胞，並且可生長在含有5-20% 胎牛血清的Leibovitz’s L-15培養基中，最適培養溫度為28-31 ℃，染色體核型均為同源染色體，染色體數目分佈以48居多，並且皆已穩定繼代40代以上。以pEGFP-N1質體DNA轉染錦鯉鰭細胞株，可觀察到綠色螢光蛋白之表現，表示細胞株有潛力利用在表現外來基因的研究。除此之外KF2細胞經過UV照射後在培養的第4小時可觀察到細胞凋亡的現象，未來也可利用於細胞凋亡的研究上。錦鯉鰭細胞株KF2細胞株對錦鯉疱疹病毒具有感受性，其感染力價可達101.5 TCID50/ml，定序後經過序列比對發現與錦鯉疱疹病毒美國株胸腺嘧啶激酶（Thymidine kinase, TK）基因相似度為100%。以上結果顯示所建立的錦鯉鰭細胞株可做為錦鯉疱疹病毒之細胞學研究材料。

Koi herpesvirus (KHV) tentatively categorized as a member of the Alloherpesviridae under the species name Cyprinid herpesvirus 3 (CyHV-3). Since 1998, lethal infections of KHV have been spreading, threatening the worldwide production of common carp and koi (both Cyprinus carpio), the first case in Taiwan has been identified in December 2002. Until now, KHV research has impeded by the lack of suitable cell line in Taiwan. In this study, two cell line were established and characterized from koi Cyprinus carpio koi fin tissue. These cell lines have been designated as Koi Fin cell 1 (KF1) and Koi Fin cell 2 (KF2), respectively. Both cell lines were epithelial-like cells. These cells multiplied well in Leibovitz’s L-15 medium, supplemented with 5 to 10% foetal bovine serum, at temperatures between 25 to 31 ℃, and have been subcultured more than 40 passages. Chromosome morphologies of KF1 and KF2 were homogeneous, and the chromosome number of KF1 and KF2 distributed major in 48. These cell lines upon transfection, using pEGFP-N1, produced significant fluorescent signals indicating their potential utility for exogenous studies. Furthermore, UV-irradiated KF2 underwent apoptosis after incubation 4 hr indicating this cell line have potential utility for apoptosis researches. KF2 was susceptible to KHV, and the infection titer can reach 101.5 TCID50/ml, and the sequence alignment is 100% similarity with KHV-USA Thymidine kinase gene. All these results suggest that the established
Koi Fin (KF) cell line is suitable for the cell biology study of Koi herpesvirus.

目錄.....................................................I
中文摘要.................................................IV
英文摘要.................................................V
圖表目錄.................................................VI
第一章、前言.............................................1
第二章、文獻整理.........................................3
2.1 魚類細胞培養.........................................3
2.1.1魚類細胞株培養的發展................................3
2.1.2魚類初級細胞培養....................................3
2.1.3魚類細胞株的應用....................................4
2.2 錦鯉疱疹病毒之簡介...................................5
2.2.1 病毒分類...........................................6
2.2.2 流行病學...........................................7
2.2.3 潛伏感染及再發.....................................8
2.2.4 診斷方式...........................................8
2.2.4.1錦鯉細胞株感受性測試..............................8
2.2.4.2聚合酶鏈鎖反應....................................9
2.2.4.3其他診斷方式......................................9
2.2.5治療方式............................................10
第三章、材料與方法 ......................................11
3.1. 錦鯉鰭細胞株之建立及其特性分析......................11
3.1.1 初級細胞培養.......................................11
3.1.2 繼代培養...........................................12
3.1.3 細胞冷凍保存.......................................13
3.1.4生長曲線分析........................................15
3.1.5染色體數目分析......................................16
3.1.6免疫螢光染色........................................18
3.1.7基因轉染............................................19
3.1.8 UV照射錦鯉鰭細胞株.................................20
3.2 錦鯉疱疹病毒之分離...................................21
3.2.1病材收集............................................21
3.2.2去氧核醣核酸之萃取..................................22
3.2.3 聚合酶鏈鎖反應.....................................23
3.2.4 瓊脂醣凝膠電泳.....................................25
3.2.5核酸定序及序列比對..................................26
3.2.6組織切片觀察........................................26
3.2.7病毒力價之測定......................................26
第四章、結果.............................................29
4.1 錦鯉鰭細胞株之建立及其特性分析.......................29
4.1.1初級細胞培養........................................29
4.1.2繼代培養............................................29
4.1.3Tubulin和Actin免疫螢光染色觀察細胞型態..............29
4.1.4不同培養條件下對細胞生長速率之影響..................30
4.1.5染色體分析..........................................31
4.1.6基因轉染............................................31
4.1.7 KF2細胞株照射UV後造成細胞凋亡現象..................31
4.2 錦鯉疱疹病毒之分離...................................32
4.2.1錦鯉疱疹病毒檢測....................................32
4.2.2病理組織切片........................................32
4.2.3 KF2細胞株對錦鯉疱疹病毒感受性測試..................32
第五章、討論.............................................34
5.1錦鯉鰭細胞株之建立及其特性分析........................34
5.1.1初級細胞培養........................................34
5.1.2不同血清添加量與細胞生長速率呈現正相關性............34
5.1.3細胞培養溫度與其生長環境有相關性....................35
5.1.4細胞基因轉染效率不佳................................35
5.2錦鯉疱疹病毒之分離....................................36
5.2.1採樣田野訪問........................................36
5.2.2採樣魚隻所分離病毒液感染細胞效率不佳................37
第六章、參考文獻.........................................39
圖一、不同代數下錦鯉鰭細胞株KF1的型態....................44
圖二、不同代數下錦鯉鰭細胞株KF2的型態....................45
圖三、錦鯉鰭細胞株KF1免疫螢光染色Tubulin 和Actin之結果...46
圖四、錦鯉鰭細胞株KF1第30代生長曲線實驗結果..............47
圖五、錦鯉鰭細胞株KF2第25代生長曲線實驗結果..............48
圖六、錦鯉鰭細胞株KF1第30代細胞染色體圖形................49
圖七、錦鯉鰭細胞株KF2第25代細胞染色體圖形................49
圖八、錦鯉鰭細胞株KF1、KF2染色體數目.....................50
圖九、 錦鯉鰭細胞株轉染pEGFP-N1之結果....................51
圖十、UV照射使錦鯉鰭細胞株KF2染色體DNA斷裂現象...........52
圖十一、台南地區罹病之食用鯉魚之外部病徵及解剖圖.........53
圖十二、 二次PCR檢測台南地區罹病食用鯉魚之結果...........54
圖十三、台南地區罹病食用鯉魚鰓部組織切片之H & E染色結果..55
圖十四、 錦鯉鰭細胞株KF2感染KHV病毒液後之細胞病變效應....56
圖十五、 錦鯉鰭細胞株KF2感染KHV病毒液後之細胞病變效應....57
圖十六、錦鯉鰭細胞株KF2經KHV病毒液感染後之PCR檢測結果....58
圖十七、KHV-TK 核酸片段序列比對結果......................59
表一、KF2細胞感染不同KHV劑量之細胞病變效應 ..............60

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