本研究以試管內、注射及飼料添加三方式探討三種分子量大小不同的幾丁聚醣 (chitosan、polyglucosamine、N-acetyl-chitooligosaccharides) 對馬拉巴石斑魚 (Epinephelus malabaricus) 免疫能力的影響。在試管內試驗，三種聚醣以12種濃度分別對馬拉巴石斑魚進行刺激頭腎吞噬細胞呼吸爆發活性測定，結果發現N-acetyl-chitooligosaccharides較其它兩種幾丁聚醣顯著刺激活性，不管添加種類為何，而除少數例外，呼吸爆發能力隨使用幾丁聚醣劑量增加而降低。注射實驗中以三種幾丁聚醣0 µg/g、10 µg/g、50 µg/g、100 µg/g腹腔注射，N-acetyl-chitooligosaccharides刺激呼吸爆發活性顯著高於其它兩種聚醣，而N-acetyl-chitooligosaccharides 10 µg/g 注射組呼吸爆發活性為各組最高，並且在0-17天時間序列觀察中較控制組能提早活化NBT反應及增進替代性補體能力，其反應時間與葡聚醣相似。飼料添加實驗則以商用鰻粉為基礎飼料，分別添加N-acetyl-chitooligosaccharides 0、0.5、1、1.5及2 g/100g diet，馬拉巴石斑魚 (平均體重120 g) 經7週餵飼不同N-acetyl-chitooligosaccharids含量飼料，魚體增重率間並沒有顯著的差異，顯示幾丁寡醣對馬拉巴石斑魚成長不會有不良的影響，飼料幾丁寡醣含量對呼吸爆發活性、替代性補體反應、凝集、溶菌酵素能力及超氧歧化酵素活性均無顯著 (P＞0.05) 影響，唯添加量為1 g/100g diet 組各項免疫生理反應似乎有偏低的現象。

This research determined the in vitro, intraperitoneal injection and dietary immunostimulatory effects on the grouper Epinephelus malabaricus of chitosan and its derivatives with different molecular weight, chitosan, polyglucosamine and N-acetyl-chitooligosaccharides. Respiratory burst activity of head-kidney phagocytes isolated from the grouper incubated in vitro with the chitosans at a range of concentrations was studied. Respiratory burst activity generally decreased with increasing dosage of chitosan products. N-acetyl-chitooligosaccharides were significantly more potent in enhancing respirtatory burst activity than the other two chitosans. Respiratory burst activity of head-kidney phagocytes of the grouper injected with three kinds of chitosans at 4 dosages was assayed. N-acetyl-chitooligosaccharides caused significantly higher respiratory burst activity than the other two chitosans. N-acetyl-chitooligosaccharides at the dosage of 10 µg/g was found to enhance the highest respiratory burst activity among treatments. In the time series assay with intraperitoneal injection by N-acetyl-chitooligosaccharides at a dosage of 10 µg/g, it was found that enhancement of NBT reduction occurred early in the time course of the study and is similar to the time series response of the glucan treatment. When the groupers (120g) were fed with diets containing 5 concentrations of N-acetyl-chitooligosaccharides including 0, 0.5, 1, 1.5, and 2 g/100g and stocked in indoor closed recirculation systems for 7 weeks, weight gain of the fish was not significantly affected by the dietary treatments. The immune status measured by respiratory burst activity, alternative complement pathway, agglutination titer, lysozyme activity and superoxide dismutase activity was not significantly affected by N-acetyl-chitooligosaccharides supplement. But feeding the grouper with N-acetyl-chitooligosaccharides at 1 g/100g diet seems to lower the immunity of the fish, although the effects were statistically insignificant.

目錄
章次 頁數
中文摘要…………………………………………………………………2
英文摘要…………………………………………………………………3
目錄………………………………………………………………………5
表目錄……………………………………………………………………6
圖目錄……………………………………………………………………8
文獻回顧………………………………………………………………....9
前言……………………………………………………………………..25
材料與方法……………………………………………………………..28
結果……………………………………………………………………..44
討論……………………………………………………………………..63
參考文獻………………………………………………………………..69

表目錄

表次 頁數
表一、飼料添加幾丁聚醣之飼料配方……………………………..………..32
表二、飼料添加幾丁聚醣實驗之一般成分分析…………………..………..33
表三、不同劑量的chitosan (C1)、polyglucosamine (C2)、N – acetyl - chitooligosaccharides (C3) 試管內刺激馬拉巴石斑魚頭腎巨噬細胞後超氧陰離子產生量…………………………….……………...….47
表四、不同劑量的chitosan (C1)、polyglucosamine (C2)、N-acetyl-chitooligosaccharides (C3) 腹腔注射馬拉巴石斑魚3天後，其頭腎巨噬細胞及經β-1,3-葡聚醣-蛋白質-脂質複合物刺激後產生超氧陰離子之活性……………………………………....49
表五、馬拉巴石斑魚腹腔注射生理食鹽水、葡聚醣及幾丁寡醣 (N-acetyl-chitooligosaccharides, C3) 後，與頭腎巨噬細胞及經β-1,3-葡聚醣-蛋白質-脂質複合物刺激後產生超氧陰離子之刺激活性隨時間序列的變化…………………………………………….….51
表六、馬拉巴石斑魚腹腔注射生理食鹽水、葡聚醣及幾丁寡醣 (N-acetyl-chitooligosaccharides, C3) 後之替代性補體活性時間序列的變化………………………………………………………….…….53
表七、馬拉巴石斑魚餵食不同幾丁寡醣添加量的飼料七週後之增重率、飼料轉換率及肝體比………………………………………….……….55
表八、餵食不同幾丁寡醣添加量的飼料七週後馬拉巴石斑魚之體組成…56
表九、馬拉巴石斑魚餵食不同幾丁寡醣添加量的飼料7週後，其頭腎巨噬細胞及經β-1,3-葡聚醣-蛋白質-脂質複合物刺激後超氧陰離子產生量…………………………………………………………………..58

表十、餵食不同幾丁寡醣添加量的飼料7週後馬巴石斑魚替代性補體活性與凝集力價…………………………………………………………..59
表十一、餵食不同幾丁寡醣添加量的飼料7週後馬巴石斑魚血清溶菌酵素活性與紅血球超氧歧化酵素活性………………………………...61

圖目錄

圖次 頁數
圖一、幾丁質及幾丁聚醣的分子結構……………………………………….14
圖二、吞噬細胞的殺菌機制………………………………………………….22
圖三、不同濃度下以chitosan (C1)、polyglucosamine (C2)、N-acetyl-chitooligosaccharides (C3) 試管內刺激馬拉巴石斑魚頭腎巨噬細胞後超氧陰離子產生量………………………………….………………………………..48
圖四、馬拉巴石斑魚腹腔注射不同劑量的chitosan (C1)、polyglucosamine (C2)、N-acetyl-chitooligosaccharides (C3) 處理3天後，與頭腎巨噬細胞經β-1,3-葡聚醣-蛋白質-脂質複合物刺激產生超氧陰離子的關係………………………………………………………………………50
圖五、馬拉巴石斑魚腹腔注射生理食鹽水、葡聚醣及幾丁寡醣 (N-acetyl-chitooligosaccharides, C3) 後，與頭腎巨噬細胞經β-1,3-葡聚醣-蛋白質-脂質複合物刺激後產生超氧陰離子產量之時間序列的關係……………………………………………………………..52
圖六、馬拉巴石斑魚腹腔注射生理食鹽水、葡聚醣及幾丁寡醣 (N-acetyl-chitooligosaccharides, C3) 後之替代性補體活性與時間序列的關係……………………………………………………………..54
圖七、馬拉巴石斑魚餵食不同幾丁寡醣添加量的飼料7週後之成長曲線..57
圖八、餵食不同幾丁寡醣添加量的飼料7週後馬拉巴石斑魚巨噬細胞超氧陰離子產生量、血清替代性補體活性及凝集力價…………………60
圖九、餵食不同幾丁寡醣添加量的飼料7週後馬拉巴石斑魚血清溶菌酵素活性及紅血球超氧歧化酵素的關係……………………………….62

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