陳秀男臺灣大學:漁業科學研究所莊柏彥Chuang, Po-YenPo-YenChuang2010-05-102018-07-062010-05-102018-07-062008U0001-2807200812012800http://ntur.lib.ntu.edu.tw//handle/246246/181683本研究從裂褶菌 (Schizophyllum commune) 發酵液中分離純化出免疫激活及抗腫瘤增殖功能之蛋白質BCⅡ。裂褶菌發酵液經硫酸銨沉澱、透析復溶後，再利用快速蛋白質液相層析系統 (FPLC) 以HIC疏水性層析、Mono-Q陰離子交換層析和Superdex 75膠體過濾層析後得到純化蛋白質BCⅡ。以SDS-PAGE蛋白質電泳和膠體過濾分析後，推估BCⅡ為一單體，分子量約為30 kDa，且由periodic acid / Schiff reagents染色後發現BCⅡ不為醣蛋白。藉由MTT assay發現到BCⅡ在濃度50、25 μg/ml時，具抑制小鼠肺癌細胞 (LLC)增殖作用；並且BCⅡ對小鼠脾細胞無細胞毒性。在免疫激活試驗方面，濃度5 μg/ml BCⅡ可直接促進自然殺手細胞 (NK cell) 之毒殺活性；不同濃度之BCⅡ亦可刺激巨噬細胞RAW 264.7產生一氧化氮 (nitric oxide) 和TNF-α。一氧化氮之產生有劑量依賴性的現象 (dose-dependent)；TNF-α則是於25 μg/ml BCⅡ時，有最高的產生量。綜合本實驗之結果，裂褶菌蛋白BCⅡ具有免疫激活之特性，並具有抗腫瘤增殖之活性，有潛力在臨床藥學上發展及應用。From the fermentation liquid of the mushroom Schizophyllum commune, a monomer protein, BCⅡ, with immunostimulative and antitumor activities had been isolated. The purification procedure was involved (NH4)2SO4 precipitation, hydrophobic interaction chromatography on Phenyl sepharose, ion exchange chromatography on Mono-Q and gel filtration on Superdex 75 by fast protein liquid chromatography (FPLC). The molecular mass of BCⅡ was 30 kDa as estimated via SDS-PAGE and gel filtration. Further staining using the periodic acid / Schiff reagents showed that BCⅡ was not a glycoprotein. BCⅡshowed antiproliferative activity toward mouse Lewis lung carcinoma (LLC) at BCⅡ concentration of 50, 25 μg/ml, respectively. And showed no cytotoxicity to mouse spleen cells by MTT assay. The immunostumulative activities of BCⅡ was also demonstrated in this study, which activate directly cytotoxicity of nature killer (NK) cell and macrophage RAW 264.7 to produce nitric oxide and TNF-α. BCⅡ stimulated macrophage RAW 264.7 to produce nitric oxide in a dose-dependent effect. The highest level of TNF-α was exhibited at BCⅡ concentration of 25μg/ml. Based on the results obtained in the present study, BCⅡ is a potent immune stimulant and could develop potentially using in medicine field.中文摘要 ………………………………………………………………..1文摘要 ………………………………………………………………..2一章 研究背景與動機 ……………………………………………...3一節 食藥用菇類的生理活性與活性成分 ……………………….4二節 裂褶菌之簡介 …………………………………………….16三節 研究動機 …………………………………………………18二章 材料方法 ……………………………………………………...19一節 裂褶菌發酵液之製備 …………………………………...19二節 裂褶菌免疫激活蛋白之純化 …………………………..20三節 裂褶菌蛋白之生化特性分析 …………………………..24四節 裂褶菌蛋白之免疫激活試驗 …………………………..29五節 數據分析 …………………………………………………37三章 結果 …………………………………………………………..39一節 裂褶菌蛋白之純化及生化特性 …………………………..39二節 免疫激活試驗 …………………………………………….42四章 討論 ………...………………………………………………...45考文獻 ………………………………………………………………48表目錄一、裂褶菌……………………………………………………………...58二、裂褶菌蛋白純化流程……………………………………………...59三、裂褶菌粗蛋白萃取液經由HIC疏水性層析管柱分離圖………...60四、裂褶菌粗蛋白萃取液經由HIC疏水性層析後SDS-電泳圖……..61五、以MTT assay測定裂褶菌蛋白對小鼠肺癌細胞 (LLC) 生長抑制之影響…………………………………………………………….62六、以MTT assay測定裂褶菌蛋白對小鼠脾細胞之細胞毒性………63七、裂褶菌蛋白萃取液經由Mono-Q陰離子交換管柱層析圖………64八、裂褶菌蛋白萃取液經由Sephadex 75膠體過濾管柱層析圖…….65九、裂褶菌蛋白BCⅡ之SDS-PAGE蛋白質電泳圖………………….66十、以Sephadex 75膠體過濾管柱層析估算裂褶菌蛋白BCⅡ之分子量………………………………………………………………….67十一、裂褶菌蛋白BCⅡ之醣蛋白染色……………………………….68十二、BCⅡ對小鼠肺癌細胞 (LLC) 生長抑制之影響……………...69十三、BCⅡ對小鼠脾細胞之細胞毒性影響…………………………..70十四、BCⅡ對自然殺手細胞毒殺作用之影響………………………..71十五、BCⅡ刺激巨噬細胞活化產生NO之比較……………………...72十六、BCⅡ刺激巨噬細胞活化產生TNF-α之比較…………………..73application/pdf2343598 bytesapplication/pdfen-US菇蕈蛋白純化抗腫瘤免疫mushroom proteinpurificationatitumorimmunostimulativehttp://ntur.lib.ntu.edu.tw/bitstream/246246/181683/1/ntu-97-R95b45013-1.pdf