臺灣博碩士論文加值系統

n-3 多元不飽和脂肪酸（PUFA）對人類與養殖⿂魚類健康相當重要，需由食
物中提供不能⾃自⾏行合成，飲⾷食中n-3 PUFA 主要來⾃自深海⿂魚油，由於來源減
少價格提升，許多研究指出具有抗發炎和增強免疫的效果，但其機制仍不
清楚，⽽而本實驗在斑⾺馬⿂魚模式動物上利⽤用腸專⼀一性啟動⼦子透過基因轉殖技
術同時增加Δ5 和Δ6 desaturase 的表現，已經由PCR 和西⽅方點墨的確認，
並增加EPA2.3 倍和DHA1.5 倍，利⽤用IP ⽅方式感染Streptococcus iniae 引起
炎症，結果顯⽰示透過表現Δ5 和Δ6 desaturase 可減少 TLR3 宿主免疫反應，
進⽽而抑制下游MYD88 誘導的炎症相關基因表現，例如：NFκB，TNFα，⽽而
使Δ5 和Δ6 desaturase 雙基因轉殖斑⾺馬⿂魚存活率相對上升20%，除此之外
我們試圖使⽤用天然具抗氧化功能的樟芝蟲草萃取液減緩傷害，在10-3 倍率
的稀釋下可以看到減緩NNV 所造成的病徵，在將來希望可以在水產養殖上
減少NNV 所造成的經濟損失。︒｡

The n-3 PUFA is important for human health, but can not be synthesised these
essential fatty acid in human body. It’s well known that n-3 fatty acids in
dietary fish oil are reported to have anti-inflammatory and injury effects in
humans, but the biochemical basis and the molecular mechanism for these
beneficial health effects is still not well understood. In our results,
Streptococcus iniae can alter the host immune response through toll-like
receptors (TLRs), and then promote the downstream target, Myd88 to induce
the expression of inflammatory related genes, and cause high mortality. By
comparison, the surrvival rate of transgene Δ5 and Δ6 desaturase zebrafish is
higher than wild type 1.33 folds. By the way, we try to use PD1 as a novel
inhibitor for NNV replication. The survival rate of PD1 treated GF1 was similar
to the control. ROS level significantly when comparing to the control cells. The
PD1 treated cells also decreased the cytopathic effect(CPE) at 42 hpi, but the
results is not so clear. We also machinery, and to understand the role of ROS
during NNV infection.

謝誌 1
縮寫表格 2
ABSTRACT 3
摘要 4
目錄 5
圖目錄 8
附錄目錄 10
第一章、前言 11
1-1多元不飽和脂肪酸介紹 11
1-2 N-3長鏈多元不飽和脂肪酸的重要性 11
1-3 N-3和N-6長鏈多元不飽和脂肪酸飲食比例的影響 13
1-4 多元不飽和脂肪酸的來源和面對的問題 14
1-5 多元不飽和脂肪酸生物合成路徑 17
1-6 ROS和發炎的關係 18
1-7 長鏈多元不飽和脂肪酸和ROS的關係 19
1-8 STREPTOCOCCUS INIAE 21
1-9 神經壞死病毒（NERVOUS NECROSIS VIRUS ） 24
1-10 研究目的 26
第二章、實驗材料 28
2-1 模式動物
ZEBRAFISH (DANIO RERIO) AB STRAIN (邰港科技公司、中研院斑馬魚中心、自家繁殖) 28
2-2 載體 (VECTOR) 28
2-3 宿主 28
2-4 培養基 28
2-5 套組 (KIT)
 29
2-6 MARKER 30
2-7 安全 DNA 內染劑
SAFEVIEWTM NUCLEIC ACID STAINS (ABM, 太鼎生技) 30
2-8 實驗藥品
 30
2-9 溶液
 32
2-10 抗體與轉漬膜 ※PRIMARY ANTIBODIES 32
2-11 儀器設備 33
第三章、材料方法 34
3-1 斑馬魚飼養與交配產卵 34
3-2 斑馬魚胚胎和組織 TOTAL RNA EXTRACT 34
3-3 DNA 電泳 36
3-4 抽取微量質體 36
3-5 確認質體 37
3-6 DNA定序 37
3-7 MICROINJECTION 38
3-8 確認腸道專一性過表現DESATURASE DELTA 5和DESATURASE DELTA 6雙基因轉殖斑馬魚 39
3-9 細胞內分子氧化程度測定 43
3-10.脂肪酸的測定 44
3-11 STREPTOCOCCUS INIAE 46
第四章 結果 48
4-1表現 DESATURASE DELTA 5和DESATURASE DELTA 6轉基因斑馬魚的驗證 48
4-2表現 DESATURASE DELTA 5和DESATURASE DELTA 6轉基因斑馬魚多元不飽和脂肪酸的含量 50
4-3表現 DESATURASE DELTA 5和DESATURASE DELTA 6轉基因斑馬魚處理感染 STREPTOCOCCUS INIAE後的存活率 51
4-4表現 DESATURASE DELTA 5和DESATURASE DELTA 6轉基因斑馬魚處理感染 STREPTOCOCCUS INIAE後腸內道菌的清除率 52
4-5表現 DESATURASE DELTA 5和DESATURASE DELTA 6轉基因斑馬魚處理感染 STREPTOCOCCUS INIAE後對免疫反應的影響 53
4-6表現 DESATURASE DELTA 5和DESATURASE DELTA 6轉基因斑馬魚處理感染 STREPTOCOCCUS INIAE後對抗氧化相關基因表現量的影響 54
4-7脂質代謝產物PD1抑制活性氧的產生，改善神經壞死病毒感染細胞病變效應 56
第五章 討論 57
5-1表現 DESATURASE DELTA 5和DESATURASE DELTA 6轉基因斑馬魚多元不飽和脂肪酸的含量 57
5-2表現 DESATURASE DELTA 5和DESATURASE DELTA 6轉基因斑馬魚處理感染 STREPTOCOCCUS INIAE後的存活率 60
5-3過度表現 DESATURASE DELTA 5和DESATURASE DELTA 6轉基因斑馬魚處理感染 STREPTOCOCCUS INIAE後腸內道菌的清除率 61
5-4過度表現 DESATURASE DELTA 5和DESATURASE DELTA 6轉基因斑馬魚處理感染 STREPTOCOCCUS INIAE後對免疫反應的影響 63
5-5過度表現 DESATURASE DELTA 5和DESATURASE DELTA 6轉基因斑馬魚處理感染 STREPTOCOCCUS INIAE後對抗氧化相關基因表現量的影響 65
5-6脂質代謝產物PD1抑制活性氧的產生，改善神經壞死病毒感染細胞病變效應 67
第六章、參考文獻 68
第七章、圖表 74
第八章、附錄 89




圖目錄
Figur 1. The construct map of transgene Δ5 and Δ6 desaturase. 74
Figur 2. TransgeneΔ5 and Δ6 desaturase was determined from F2 Δ5 and Δ6 desaturase transgenic fish.
(A) Delta-5 and delta-6 desaturase transgenic zebrafish.(B) Genomic DNA PCR was used to determine the transgenes zebrafish F1 in genomic DNA. Line 1: Elovl transgene zebrafish, primer：Elovl; Lane 2：triple transgene zebrafish, primer：Elovl; Lane 3：triple transgene zebrafish, primer：fad5/fad6; Lane 4：Elovl transgene zebrafish, primer：EF1α, Lane 5：triple transgene zebrafish, primer：EF1α (C) cDNA PCR was used to determine the transgenes zebrafish F1 in cDNA. Line 1: Elovl transgene zebrafish, primer：Elovl; Lane 2：Elovl transgene zebrafish, primer：fad5/fad6; Lane 3：Elovl transgene zebrafish, primer：EF1α; Lane 4：triple transgene zebrafish, primer：Elovl, Lane 5：triple transgene zebrafish, primer：fad5/fad6; Lane 6：triple transgene zebrafish, primer：EF1α(D)Half-quantitation of cDNA PCR was used to determine the transgenes zebrafish F1 in cDNA. The expression of EF-1α served as an internal control. (E) Realtime-PCR analysis of genomic DNA PCR was used to determine the transgenes zebrafish F1 in genomic DNA. The expression of EF-1α served as an internal control. (F) Realtime-PCR analysis of cDNA PCR was used to determine the transgenes zebrafish F2 in cDNA. The expression of EF-1α served as an internal control. (G) Western blot against the V5-tag fused to delta-5 and delta-6 desaturase in wild-type and transgenic fish. The expression of β-actin served as an internal control. (H) Half-quantitation of western blot against the V5-tag fused to delta-5 and delta-6 desaturase in wild-type and transgenic fish. 77
Figur 3. Total PUFAs in transgenic and wt zebrafish 79
Figur 4. Survival rate of zebrafish after infected with Streptococcus iniae in 36 hours
. 79
Figur 5. Bacteria contents in zebrafsih after Streptococcus iniae infection. Tissues were collected and culture on BHI agar plates for counting the Bacteria content (CFU/mL) after 24 hour culture . 79
Figur 6. Inflammatory gene expression in wild-type and transgenic F2 delta-5 and delta-6 desaturase fish challenged with Streptococcus iniae. (A)Cytokines (B)COX2 (C)TLR3(D)Myd88 81
Figur 7. Anti-oxidase gene expression in wild-type and transgenic F2 delta-5 and delta-6 desaturase fish challenged with Streptococcus iniae. (A)Prx2 (B)Prx6 (C)exSOD (D)CuSOD (E)MnSOD (F)Catalase 84
Figur 8. The morphology of NNV infection with multiplicity of infection (MOI) dose dependent in GF-1 cells. 85
Figur 9.The morphology of NNV infected with MOI o.1 in GF-1 cells. A: The PD1 treating was before the virus infected. B: The PD1 treating and the virus infection were the same -me. C: The PD1 treating was after the virus infected. 86
Figur 10. The survival rate of the different treatments with the NNV infected in GF1 cell. 87
Figur 11.The effect of an-virus drug treatment, Antrodia cinnamomea liquid-liquid extraction, with different concentration to NNV infected in GF1 cell. (A) the morphology (B) cytopethic effect 88














附錄目錄
附錄 一 89
附錄 二 89
附錄 三 90
附錄 四 91
附錄 五 92
附錄 六 95
附錄 七 98
附錄 八 100
附錄 九 103
附錄 十 104
附錄 十一 105

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