臺灣博碩士論文加值系統

本實驗室利用動物細胞培養於無血清的狀態下找尋食品中的有效成分進而進行動物試驗。於此一研究中，於九種水產魚類中，篩選旗魚、虱目魚、吳郭魚、鯖魚、黃鰭鮪、鱸魚、正鰹、鰻魚、秋刀魚，熱水萃取的方式所取得之熱水萃取物可使人類B細胞株HB4C5細胞增生，取吳郭魚及虱目魚；本實驗室發現九孔，具有生理活性物質。取吳郭魚、虱目魚及九孔等之新鮮材料均質，以100℃熱水萃取30分鐘，離心去除顆粒即以減壓濃縮機濃縮將熱水萃取物以硫酸銨百分濃度0-50%分劃，透析、溶液凍乾。虱目魚、吳郭魚熱水萃取物經硫酸銨百分濃度0-50%分劃之蛋白質進行動物細胞分析，發現吳郭魚及虱目魚的熱水萃取物對人類B細胞株HB4C5、及人類骨髓細胞株K562具有刺激增生的效果。九孔熱水萃取物進行免疫功能的評估，將九孔去除殼及內臟，均質、以100℃熱水萃取30分鐘對人類B細胞株HB4C5，或人類骨髓細胞株HL60，具刺激細胞增生的生理活性物質。混合吳郭魚、虱目魚熱水萃取物經硫酸銨百分濃度0-50% 所沉澱下的蛋白質及九孔熱水萃取物等比例混合，將之命名為三合一蛋白質，進行動物試驗。動物試驗主要探討兩個議題，細胞性免疫力及體液性免疫力。餵食餌料內含三合一蛋白質，劑量佔小鼠每天攝食飼料的1% 及2% ，為期八週實驗，犧牲小鼠取其脾臟細胞及腹腔細胞以裂殖Con A、PHA及LPS刺激，發現於非特異性實驗組，其T細胞增生與控制組比較起來，於統計上具有顯著的差異，B細胞的表現與控制組比較起來具有兩倍以上的差異，腹腔中巨噬細胞佔大多數，經餵食三合一蛋白質後可刺激巨噬細胞增生；特異性組方面，脾臟細胞及腹腔細胞經裂殖原的刺激下與控制組比較起來於統計上具有差異；經動物實驗，三合一蛋白質可促進細胞性免疫力之增強。體液性免疫力於特異性組及非特異性組，沒有顯著促進抗體分泌；於小鼠週邊血液的吞噬能力來說，餵食三合一蛋白質組之BALB/c雌鼠，於非特異性組可增強吞噬比例及能力；特異性組則不具相同的結果，在增強吞噬比例及能力之促進效果就不顯著。

In search of the growth and the immunity promoting substance from food components. We used mammalian cell lines with serum-free medium under animal experiment model. We reported here with the growth and the immunity promoting substances from fish species. Fresh material were homogenized and then boiled at 100℃ for 30 mins. After centrifugation, the supernatant was further concentrated by vacuum evaporation. Through 30%-50% ammonium sulfate saturation precipitation (WG30-50). Those dialysis (ultrafiltration M.W.10 Da. cut off) were lyophilized. The result showed that extracts of Tilapia, milk fish enhanced cell proliferation toward human-human hybridoma cell line HB4C5 and enhanced cell proliferation toward human marrow cell lines HL60. Furtheμrmore, Taiwan small abalone, Halitotis diversicolor was selected for immunological function assessment. After small abalone was removed from the viscera, the pulp was treated by hot water (100℃, 30 mins). Hot-water extracts promoted cell proliferation toward human hybridoma cell line HB4C5, as well as macrophage-like cell lines (U937, UMf, THP-1 and J774.1) . We mixed Tilapia WGT3050, milk fish WGM3050 and small abalone, which show the immune bioactivity and named three-in-one. BALB/c mice were fed with different dose of three-in-one. One and two percent of mice daily eating were supplied by the three-in-one. Three-in-one significantly increased almost three-fold proliferation of spleen cell which is stimulating with LPS, either in specific or non-specific group is acceptable, and be stimulating with PHA in specific experiment. It increased one-fold to three-fold proliferation of PEC which is stimulating with PHA and LPS in specific or non-specific animal experiment. Two percent of mice daily eating is supplied by three-in-one also significantly increased in secretion of antibody IgG in specific or non-specific group. Both of the different dose were fed mouce also increased the ability of phagocytosis in whole blood. Through Phenyl column and Amicom ultrafiltration, we purify bioactive substances from WGT3050. These results obtained in this study indicated that the extract of three in one should be good candidate for health food development.

中文摘要 VI
英文摘要 VIII
壹、 前言 1
貳、 文獻整理 4
一、 水產資源之重要性 4
二、 台灣養殖九孔、吳郭魚、虱目魚之簡介 5
1. 九孔 5
2. 吳郭魚 6
3. 虱目魚 7
三、 水產品之免疫調節生理功能研究 9
1. 水產貝類之研究 9
2. 海藻類之研究 10
四、 動物之免疫調節系統 10
1. 細胞性免疫反應 10
2. 體液性免疫反應 13
3. 巨噬細胞於免疫之調節功能 14
參、 材料與方法 16
一、 實驗大綱 16
二、 實驗材料 20
1. 細胞株 20
2. 九孔、吳郭魚及虱目魚之材料來源 20
3. 藥品 20
3-1. 吳郭魚及虱目魚熱水萃取物蛋白質之分劃 20
3-2. 動物細胞培養 21
3-3. 蛋白質定量 21
3-4. 細胞存活率之測定 21
3-5. 九孔、吳郭魚和虱目魚增強免疫因子之純化 21
3-6. 動物實驗 22
3-7. BALB/c小鼠血清中抗體測定 23
3-8. 西方點墨分析法 23
4. 設備 23
三、 實驗方法 25
1. 懸浮性細胞之培養 25
2. 九孔、吳郭魚及虱目魚熱水萃取物之製備 25
2-1. 九孔熱水萃取物的製備 25
2-2. 吳郭魚熱水萃取物的製備 25
2-3. 虱目魚熱水萃取物的製備 26
3. 蛋白質定量 26
4. 細胞存活率 27
5. 吳郭魚熱水萃取物中免疫活性因子之純化 27
5-1. Sephadex G-100管柱層析 27
5-2. Phenyl-Toyopearl管柱層析 27
6. 膠體電泳分析 28
6-1. Stock solutions之配製 28
6-2. 膠體配製 29
6-3. 樣品製備及操作方法 29
6-4. 膠片染色與脫色 30
6-5. 膠片的乾燥 30
7. 長時間餵食動物免疫試驗 30
7-1. 材料餵食與餵食方法 30
a. 三合一飼料配方 30
b. 三合一飼料製程 31
7-2. 實驗動物 31
7-3. 特異性免疫反應 32
7-4. 血樣的收集 32
7-5. 脾臟及腹腔細胞之分離 32
a. 脾臟細胞之分離 32
b. 腹腔細胞之分離 33
7-6. 細胞性免疫分析 33
a. 修飾 MTT 法 33
7-7. 抗體分泌實驗 34
a. 非特異性抗體IgG之測量 34
b. 特異性抗體IgG之測量 34
7-8. 吞噬細胞活性 35
8. 吳郭魚熱水萃取物免疫活化因子之純化 35
9. 西方點墨法 (Western blotting) 35
肆、 結果與討論 37
一、 九孔、吳郭魚及虱目魚熱水萃取物之製備 37
1. 九孔原料製備 37
2. 吳郭魚原料之製備 37
3. 虱目魚原料之製備 37
4. 吳郭魚及虱目魚熱水萃取，去除油脂的方法 37
二、 熱水萃物經硫酸銨競爭與水結合，所沉澱之蛋白質對細胞之影響 38
三、 九孔、吳郭魚及虱目魚熱水萃取物，對動物體內免疫調節功能影響 39
1. 體重之變化 40
2. 攝食利用率 40
3. 器官重 41
4. 抗體生成量 41
四、 BALB/c小鼠攝食三合一蛋白質，對脾臟細胞及腹腔細胞增生影響 41
五、 小鼠週邊血細胞吞噬能力測量 42
六、 吳郭魚熱水萃取物免疫生理活性物質之純化 43
七、 產品的品質管控 43
伍、 結論 45
陸、 參考文獻 46
柒、 圖表 錯誤! 尚未定義書籤。

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