臺灣博碩士論文加值系統

神經壞死病毒(Nervous necrosis virus,NNV)感染魚種廣泛，在台灣引起許多種海水養殖魚苗的高死亡率包括近年熱門養殖魚種—石斑魚，因此針對病毒防治之藥物開發顯得非常重要。本論文選擇紫錐菊（Echinacea purpurea）萃取物（EPE），以石斑魚細胞株為探討紫錐菊抗NNV的模式，先是利用WST-1檢測紫錐菊粹取物的細胞毒性，在不具細胞毒性濃度200μg/ml以下來進行攻毒試驗(M.O.I = 1)。將攻毒實驗所收集到的細胞使用即時定量聚合酶連鎖反應來偵測(1)細胞內的病毒量(2)促發炎基因的腫瘤壞死因子α跟介白素-1β (3)干擾素(Interferon,IFN)、Mx，來證明紫錐菊是否有抗神經壞死病毒的可能性。在本次研究的結果中，在含有紫錐菊持續培養的攻毒組別可以在48小時內的病毒量顯著低於攻毒後未含有紫錐菊的組別，基因的表現只能說明受NNV感染後有促使這些基因的表現，含紫錐菊培養的攻毒組別的基因表現量並沒有因紫錐菊而有所誘發，推測紫錐菊萃取物是具有抑制神經壞死病毒可能性。

Nervous necrosis virus (NNV) can infect more fish that has caused a high mortality rate of numerous marine fish species including grouper at larval stage in Taiwan. Thus, it is important to develop an anti-NNV drug to prevent virus infection and outbreak.In this thesis, Echinacea purpurea extract (EPE) was selected as a model to investigate anti-NNV effect on grouper cell line. WST-1 assay was used to determine the cytotoxicity in grouper cell line and the suitable concentration of EPE was found to be less than 200μg/ml. The different concentrations of EPE which are less than 200μg/ml were selected for the NNV challenge experiment (M.O.I =1) in grouper cell line. The cells were collected and real-time quantitative PCR was used to quantify the (1) viral titer in grouper cell, (2) pro-inflammatory genes such as tumor necrosis factor-α and interleukin-1β, (3) interferon &; Mx gene, to verify the anti-NNV effect of EPE extract.In our results showed that the NNV viral titers were significantly decreased in the EPE treated groups compared with the non-EPE treated groups within 48 hours. But in gene expression not significantly between EPE treated and non-EPE treated that expression only prove non-EPE treated NNV infect grouper brain cell.Speculated ous study experiment Echinacea extract was possibility inhibited NNV .

摘要……………………………………………………………………………………I
Abstract……………………………………………………………………………II
目次…………………………………………………………………………………Ⅲ
圖目次……………………………………………………………………………Ⅳ
表目次………………………………………………………………………………Ⅴ
第一章 緒論………………………………………………………………………1
第一節 台灣石斑魚養殖…………………………………………………………1
第二節 神經壞死病毒介紹………………………………………………………3
第三節 魚類免疫系統概述………………………………………………………5
第四節 中草藥於水產養殖應用…………………………………………………6
第五節 紫錐菊（Echinacea purpurea）概述…………………………………7
第六節 研究目的…………………………………………………………………7
第二章實驗材料……………………………………………………………………9
第三章實驗方法……………………………………………………………………11
第一節 神經壞死病毒生產與感染石斑魚腦細胞…………………………11
第二節 GF-1與石斑魚腦細胞株繼代培養…………………………………11
第三節 病毒力價計算………………………………………………………12
第四節 紫錐菊培養液製備…………………………………………………12
第五節 WST-1細胞存活率測試……………………………………………12
第六節 不同濃度下的紫錐菊抗病性測試…………………………………12
第七節 total RNA萃取………………………………………………………13
第八節 聚合酶連鎖反應……………………………………………………13
第九節 紫錐菊萃取物(EPE)在斑馬魚苗的毒性測試……….……………… 14
第四章結果……………………………………………………………………15
第一節 神經壞死病毒之分離與感染石斑魚腦細胞…………………………15
第二節 不同濃度紫錐菊對於魚類細胞的存活率……………………………15
第三節 攻毒後在不同濃度EPE持續培養之石斑魚腦細胞病毒量…………15
第四節 攻毒後石斑魚腦細胞株的促發炎TNF-α、IL-1β基因表現量……16
第五節 攻毒後石斑魚腦細胞干擾素干擾素與Mx基因表現量……………16
第六節 紫錐菊萃取物(EPE)在斑馬魚苗的毒性測試…………………………16
第五章討論………………………………………………………………………17
第六章結論………………………………………………………………………21
第七章參考文獻…………………………………………………………………… 22
附錄…………………………………………………………………………………37

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