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研究生:施寅澤
研究生(外文):Shi, Yin-Ze
論文名稱:白蝦Litopenaeus vannamei接受馬尾藻萃取物引發之內源性物質對其先天性免疫反應
論文名稱(外文):Immune response of white shrimp Litopenaeus vannamei receiving endogenous molecules inducesd by Sargassum oligocystum extract
指導教授:陳建初陳建初引用關係
指導教授(外文):Chen, Jiann-Chu
口試委員:李安進鄭學淵劉俊宏陳建初
口試委員(外文):Lee, An-ChinCheng, Sha-YenLiou, Jiun-HungChen, Jiann-Chu
口試日期:2016-01-08
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:58
中文關鍵詞:白蝦先天性免疫力內源性分子馬尾藻萃取物溶藻弧菌
外文關鍵詞:Litopenaeus vannameiinnate immunityEM-SOESargassum oligocystum extractVibrio alginolyticus
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白蝦血球經與馬尾藻萃取物(Sargassum oligocystum extract, SOE)處理,引發內源性分子(endogenous molecules–Sargassum oligocystum extract, EM-SOE)。研究白蝦血球接受EM-SOE,觀察去顆粒化、血球大小、酚氧化酵素活性及呼吸爆。蝦血球經EM-SOE培養後,顆粒型血球去顆粒化,血球型態改變,相對大血球百分比(percentage of large cell)由86% 減少至41%,酚氧化酵素活性及呼吸爆增加。白蝦分別經注射marine saline(MS)、EM-C、SOE、EM-SOE,經1天,檢測透明球、顆粒球、總血球、酚氧化酵素活性、呼吸爆、超氧歧化酵素活性、溶菌酶活性免疫因子,研究白蝦感染溶藻弧菌3小時後,檢測對弧菌的吞噬活性、吞噬活性、清除能力,研究感染溶藻弧菌96小時後,活存率。結果發現白蝦經SOE、EM-SOE處理後,免疫因子提升,對弧菌的吞噬活性、吞噬指數、清除能力及活存率均高於MS及EM-C處理組。液相層析串聯質譜儀(LC-ESI-MS/MS)顯示EM-SOE具有損傷相關模式分子(damage-associated molecular pattern, DAMP)如antimicrobial peptides、proPO、proteinase/proteinase inhibitors、blood clotting protein、signaling transduction molecules、heat shock proteins等。證實SOE引發白蝦血球壞死,產生EM-SOE具DAMP相關蛋白質,引起顆粒型血球去顆粒化、酚氧化酵素活性及超氧陰離子增加,啟動先天性免疫力及一旦受到弧菌感染的抵抗力增加。
Sargassum oligocystum extract (SOE) incubated with white shrimp Litopenaeus vannamei hemocytes caused necrosis and released endogenous molecules, namely endogenous molecules Sargassum oligocystum extract (EM-SOE). Hemocytes incubated with EM-SOE caused degranulation, and changed percentage of large cells that deceased from 86% to 41%. Shrimp hemocytes incubated with EM-SOE caused increases in phenoloxidase (PO) activity and respiratory burst (RB). The immune parameters of white shrimp that received EM-SOE were examined after 24 h. The immune parameters including hyaline cells, granular cells, total hemocyte count (THC), PO activity, RB, superoxide dismutase (SOD) activity, and lysozyme activity increased significantly when shrimp received SOE and EM-SOE, compared to those of shrimp that received MS. Shrimp that received SOE and EM-SOE had increased phagocytic activity, phagocytic index and clearance efficiency to V. alginolyticus after 3 h. In another experiment, survival rate of shrimp that received SOE and EM-SOE after 24 h, and challenged with V. alginolyticus were significantly higher than that of control shrimp and then challenged with V. alginolyticus.A LC-ESI-MS/MS analysis indicated that EM-SOE contained damage-associated molecular patterns (DAMPs) including HMGBa, histone 2A (H2A), H2B, and H4, and other immune proteins like proPO I, Rab GPTase, and Rab 11 GPTase. In conclusion, EM-SOE induced by SOE contains DAMPs and other immune molecules, trigger innate immunity as evidences of hemocyte degranulation, change in cell size, and increases in PO activity and release of superoxide anion in vitro. Shrimp that received EM-SOE showed increases in immune parameters and increased resistance against V. alginolyticus infection.

謝辭 i
中文摘要 ii
Abstract iii
目錄 iv
表目錄 vii
圖目錄 viii
第一章 前言 1
第二章 文獻整理 4
2.1 白蝦的分類與生態習性 4
2.2 甲殼類免疫防禦 4
2.2.1 血球(或稱血淋巴球) 5
2.2.2 先天性免疫系統的辨識與啟動 5
2.2.3 原酚氧化酵素活化系統 6
2.2.4 aPeroxinectin(黏附蛋白,或稱76 kDa蛋白) 6
2.2.5 吞噬作用與抗氧化機制 7
2.2.6 結癤(nodulation)與胞膜(encapsulation) 7
2.2.7 抗菌胜肽(antimicrobial peptide) 7
2.3 免疫刺激物(immunostimulant) 8
2.4 海藻 9
2.5 海藻多醣及海藻萃取物對魚蝦免疫反應 9
2.6 內源性分子(endogenous molecules) 10
第三章 材料方法 12
3.1 實驗動物 12
3.2 實驗條件 12

3.3 馬尾藻萃取物製備 12
3.4 內源性分子(endogenous molecules, EM)溶液製備 12
3.5 弧菌(Vibrio alginolyticus)培養 13
3.6 體外(in vitro)實驗 13
3.6.1 血球去顆粒化 13
3.6.2 血球大小 13
3.6.3 血球活存 14
3.6.4 血球壞死 14
3.6.5 血球in vitro 酚氧化酵素(PO)活性 15
3.6.6 血球in vitro 呼吸爆(RB) 15
3.7 體內(in vivo)實驗 16
3.7.1 血球數 16
3.7.2 酚氧化酵素(phenoloxidase, PO) 活性 16
3.7.3 呼吸爆(respiratory burst, RB) 17
3.7.4 超氧歧化酵素(superoxidase dismutase, SOD)活性17
3.7.5 溶菌酶(lysozyme)活性 17
3.7.6 吞噬效能(phagocytic activity)與吞噬指數(phagocytic index) 18
3.7.7 清除能力(clearance efficiency) 18
3.7.7 弧菌Vibrio alginolyticus攻擊試驗 18
3.8 蛋白質凝膠電泳分析 19
3.8.1 膠體製備 19
3.8.2 樣本製備 19
3.8.3 蛋白質電泳 19
3.8.4 液相層析串聯質譜儀(LC-ESI-MS/MS)分析EM-SOE蛋白質20
3.9 統計分析 20
第四章 結果 21
4.1 體外實驗 21
4.1.1 白蝦血球去顆粒化作用 21
4.1.2 白蝦血球大小 21
4.1.3 白蝦血球活存 21
4.1.4 白蝦血球壞死 22
4.1.5 aIn vitro 酚氧化酵素活性 22
4.1.6 aIn vitro 呼吸爆 22
4.2 體內實驗研究免疫因子 22
4.2.1 血球數 23
4.2.2 酚氧化酵素活性 23
4.2.3 呼吸爆 23
4.2.4 超氧歧化酵素活性 23
4.2.5 溶菌酶活性 23
4.3 接受EM-SOE的白蝦對溶藻弧菌吞噬作用及清除能力 23
4.4 接受EM-SOE的白蝦感染溶藻弧菌後的抵抗力 24
4.5 蛋白質電泳分析 24
4.6 液相層析串聯質譜儀(LC-ESI-MS/MS)分析EM-SOE蛋白質 24
第五章 討論 25
第六章 結論 27
第七章 參考文獻 28
附錄 54

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