黃穰臺灣大學：海洋研究所李 惠 婷Lee, Hui-TingHui-TingLee2007-11-272018-06-282007-11-272018-06-282005http://ntur.lib.ntu.edu.tw//handle/246246/56616本論文是從海洋褐藻小海帶萃取多種多醣類與蛋白質，經過分離純化後對老鼠巨噬細胞（RAW 264.7）與人類T 淋巴細胞（CCRF-CEM）做體外活性檢測，以及評估其對免疫能力，如對一氧化氮（NO）、腫瘤壞死因子（tumor necrosis factor, TNF-α）及干擾素（Interferon-γ, IFN-γ）之影響。結果顯示小海帶以不同方式之萃取物均能促進此二種免疫細胞的增殖活性。小海帶褐藻酸及褐藻酸鈉多醣類，均能顯著刺激其免疫細胞的增殖，如褐藻酸鈉濃度為0.1 µgmL-1濃度下，即能促進其增殖力達60 %。小海帶以PBS萃取，並經硫酸銨沈澱、膠體過濾管柱與離子交換管柱之層析及純化後，所得之小海帶免疫活性之蛋白質（簡稱 EBP），其蛋白質的分子量為69 kDa，醣含量則佔20.37 %，屬一種醣蛋白，亦為單聚體蛋白。此醣蛋白對巨噬細胞與T淋巴細胞有顯著刺激增殖的效果，其濃度範圍為0.04∼0.25 µgmL-1。進一步以sandwich ELISA 法分析，顯示此醣蛋白能刺激免疫細胞產生多量細胞激素（cytokines），如 TNF-α、IFN-γ及NO。EBP對酸鹼值的變化亦具耐受性，在pH值2.0∼13.0範圍內均能維持很高的活性。再者，EBP於121 ℃下加熱20分鐘，仍保有原來活性的75 %，顯示EBP也具耐熱性。由本實驗得知小海帶具有免疫活性之醣蛋白與多醣類，值得進一步研究，以期未來可被應用在醫藥上。Various extracts of the marine brown alga Endarachne binghamiae were assayed in vitro for immunological activity against murine macrophages and human T cells. In addition, the immune-active substances were extracted, purified and characterized biochemically. It appeared that all extracts used, including saline, hot-water and aqueous ethanolic extracts, could significantly stimulate macrophage and T cell proliferation to various extents. Sodium alginate had greater activity than alginic acid and isopropanol-precipitated polysaccharide, with maximal activity, i.e. proliferation increased by 70~100 %, at 0.05~0.1 µgmL-1. A novel protein assigned as EBP ( Endarachne binghamiae protein) was obtained after being extracted in phosphate buffered saline, precipitated by saturated ammonium sulfate and finally purified by gel filtration and ion-exchange column chromatography. The purified protein had molecular weight of 69 kDa. It was a monomer glycoprotein, containing 20.37 % carbohydrate moiety. EBP also stimulated strongly phagocytes and lymphocytes to proliferate, with maximal proliferation, i.e. by twofold, at 0.04~0.25 µgmL-1. Further assays showed distinct increases of cytokines, i.e. TNF–α, IFN–γ, and nitric oxide, generated and released from macrophages and T cells in the cultures with EBP, using sandwich ELISA method. EBP was rather stable in immunity while subjected to 121 ℃ and a wide range of pH values (2.0~13.0). The above findings suggested that the reported alga is a promising source of proteins and polysaccharides used for immunostimulation and immuno-modulation in therapy and medicine.目 錄 頁次 中文摘要…………………………………………………………………I 英文摘要……………………………………………………………….II 圖 次………………………………………………………………….III 壹、前言……………………………………………………………...1 貳、文獻整理………………………………………………………….3 一、免疫調節活性的研究……………….…………………………3 二、多醣類與蛋白質的免疫生理活性…………………….………7 參、材料與方法………………………………………………...…..9 一、實驗流程……………..…………………………………..… 9 二、實驗材料…………….……………………………………………10 三、實驗方法……………………………………………………… 13 肆、結果…………………………………………………………..….22 一、不同採集地點之小海帶萃取液增殖活性………………… 22 二、不同萃取時間長短對小海帶PBS萃取液增殖活性的影響……22 三、不同溫度萃取對小海帶增殖活性的影響……………………22 四、不同萃取液萃取小海帶對巨噬細胞增殖之比較……………..23 五、多醣類對免疫細胞增殖的影響..........................23 六、小海帶免疫活性蛋白質之分離與純化………………………..24 七、溫度及處理時間對EBP增殖活性的影響....................25 八、不同pH值對EBP的增殖活性之影響………………….………..26 九、 EBP促進免疫細胞產生細胞激素........................26 十、 EBP對毒胞活性NO產生之影響…………………...……..……26 伍、討論………………………….....………………………………27 陸、參考文獻…………………..……………...…………………..32 附錄一………………..………………………………..…………….68 附錄二………………....……………………………..…………….69879726 bytesapplication/pdfen-US小海帶Endarachne binghamiae[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56616/1/ntu-94-R92241202-1.pdf