臺灣博碩士論文加值系統

在許多南亞國家，石斑魚被認為是一種高經濟價值的水產品。然而，因石斑魚十分容易受病毒感染，而造成水產養殖產業重大的經濟損失。在台灣，虹彩病毒(Iridovirus)是海水魚養殖產業經常面臨的主要病原。虹彩病毒不僅攻擊魚苗、稚魚，也會感染體型較大、經濟價值極高的石斑魚成魚，並導致成魚大量死亡。中草藥萃取物與傳統的免疫刺激物可望被應用於水產養殖，取代抗生素與化療藥物的使用。在此研究中，選擇三種中草藥萃取物(魚腥草、虎杖、黃耆)於石斑魚腎臟細胞株(Grouper kidney cells, GK cells)進行測試，並評估該三種中草藥萃取物對虹彩病毒的作用與影響。在實驗中，將不同濃度的魚腥草與虎杖，分別以0µg/mL(控制組)、1µg/mL、5µg/mL與10µg/mL加入受虹彩病毒感染的細胞；不同濃度的黃耆，分別以0µg/mL(控制組),1µg/mL與5µg/mL進行預處理和共處理。此外，實驗中也測定以不同濃度萃取物處理後細胞的基因表現亮，包含IgD、IgM、MHC-II、IL-1β、Mx、TNF-α、MHC-I、C3與IL-6，共九種基因。結果顯示，細胞經三種中草藥萃取物與病毒共同處理72小時後，依然可以偵測病毒的存在；而三種中草藥萃取物預處理細胞後以病毒攻擊72小時後，全部組別皆未偵測病毒的存在。在基因表現實驗，利用聚合酶鏈鎖反應 (polymerase chain reaction, PCR)偵測魚腥草萃取物處理組中IgD、IgM、 MHC-II、IL-1β與Mx的基因表現量，但未能偵測到TNF-α、MHC-I、C3與IL-6的基因表現量。魚腥草、虎杖與黃耆萃取物處理後的實驗組，TNFα基因的表現量都有微小的差異。結果顯示，虎杖與黃耆萃取物處理組都能使TNF-α表現，然而魚腥草萃取物處理組只有濃度10µg/mL組別有偵測到TNF-α表現。特別的是，黃耆萃取物濃度1µg/mL處理組有偵測到MHC的基因表現。

Groupers are known to be an economically valuable aquaculture species in Southeast Asian countries. Groupers however, are known to be prone to viral infections that have been the root cause of great economic loss for aquaculture industries. Grouper iridovirus in Taiwan, is a major pathogen in marine fish aquaculture. This viral disease not only affect fry and juvenile grouper but are also known cause deaths in market-sized grouper, which are known to be a high priced, high demand cash crop in tropical marine culture. Herbal extracts as well as more traditional immuno stimulants could be applied in fish culture to replace antibiotic and chemotherapeutic agents. In this study, examination of three Chinese herbal extracts (Houttuynia cordata, Polygonum cuspidatum and Astragalus spp.) applied on GK cells. The experiment was carried out to evaluate the effects of three herbs on GK cells to against GIV. In this experiment, GK cells infection by iridovirus was replaced at 0µg/ml (control), 1 µg/ml, 5µg/ml and 10µg/ml of two herbs (Houttuynia cordata and Polygonum cuspidatum) at 0µg/ml (control), 1 µg/ml and 5 µg/ml in treatment of Astragalus spp. in pre-treatment and co-treatment. In other experiment, GK cells were evaluated with nine gene expression of IgD, IgM, MHC-II, IL-1β, Mx, TNF-α, MHC-I, C3 and IL-6 at different dose of herbal extracts. The result showed that the virus was still detected in co-treatment GK cells after 72 hours treated with three herbal extracts while in pre-treatment, the virus was not detected in all samples of this treatment. In gene expression experiment, IgD, IgM, MHC-II, IL-1β, and Mx were detected by using PCR method in treatment with Houttuynia cordata while TNF-α, MHC-I, C3 and IL-6 were not detected in this herb. There was a minimal difference between Houttuynia cordata, Polygonum cuspidatum and Astragalus spp. treatments in TNF-α gene expression. The result indicated that TNF-α was detected in both treatments of Polygonum cuspidatum and Astragalus spp in treatment groups only. However, in Houttuynia cordata treatment, TNF-α was detected at 10µg/ml only and it was not detected in control groups. Specially, MHC was detected in Astragalus spp. at 1 ug/mL only.

ACKNOWLEDGEMENT I
CHINESE ABSTRACT II
ABSTRACT III
TABLE OF CONTENTS IV
LIST OF TABLES VI
LIST OF FIGURES VII
1. INTRODUCTION 1
2. LITERATURE REVIEW 3
2.1. Grouper aquaculture 3
2.2. Orange spotted grouper, Epinephelus coioides 4
2.3. Grouper viral diseases 5
2.4. Iridoviruses 5
2.5. Chinese Herbs 6
2.5.1. Houttuynia cordata 6
2.5.1.1. Ethnomedical uses 7
2.5.1.2. Anti-viral activity 7
2.5.1.3. Anti-bacterial activities 9
2.5.2. Astragalus (Huang Qi) 9
2.5.3. Polygonum cuspidatum (Hu Zhang) 11
3. MATERIALS AND METHODS 13
3.1. Equipment 13
3.2. Cell cultivation, passage cells and viral infection 15
3.2.1. Cell cultivation, passage cells 15
3.2.2. Preparation of Virus Inoculation 15
3.3. Transformation of bacteria cells 15
3.4. Mini preparation of plasmid DNA 16
3.5. Preparation of Chinese Herbs 16
3.6. Expression Analysis of nine selected immune-related genes in E. coioides by PCR 17
3.6.1. RNA extraction 17
3.6.2. Polymerase chain reaction detection for Expression of immune genes 17
3.7. DNA extraction and primer design for PCR in virus treatment 18
4. RESULTS 19
4.1. Cytotoxicity in vitro of herbal extracts of GK cells 19
4.2. Gene expression of IgD, IgM, MHC-II, IL-1β, Mx, TNF -α, MHC-I, C3 and IL-6 19
4.3. The effect of herb extracts on cell inoculated by iridovirus 20
4.4. Replication of GIV in GK cells (Challenge experiments with GIV) 21
5. DISCUSSION 23
REFERENCES 27
APPENDICES 34

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