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研究生:劉育靈
研究生(外文):Yu-Ling Liu
論文名稱:金針菇免疫調節蛋白 FIP-fve 生物可及性之研究
論文名稱(外文):Bioaccessibility Study for Fungal Immunomodulatory Protein FIP-fve
指導教授:許輔許輔引用關係
口試委員:周志輝吳思敬繆希椿
口試日期:2013-07-15
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:園藝暨景觀學系
學門:農業科學學門
學類:園藝學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:67
中文關鍵詞:FIP-fve生物可及性體外模擬腸胃道消化腸繫膜淋巴結培耶氏斑
外文關鍵詞:FIP-fvebioaccessibilityin vitro gastrointestinal digestionmesenteric lymph nodesPeyer’s patch
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FIP-fve 為純化自金針菇之免疫調節蛋白,研究指出 FIP-fve 可刺激人類周邊血淋巴細胞增生及分泌 IFN-γ,並使免疫反應趨向於 TH1 反應;體內試驗亦發現,口服 FIP-fve 可降低小鼠食物過敏的症狀、延長罹癌小鼠之壽命及抑制腫瘤大小。生物可及性 (bioaccessibility) 是指活性成分自食物基質釋出後,可通過腸胃道的部分,用來評估活性成分的可利用性,其大多採取體外模擬腸胃道消化的試驗來證明。本實驗欲探討金針菇免疫調節蛋白 FIP-fve 經模擬腸胃道消化後之穩定性,並評估其是否具有活化腸道免疫細胞之功能。第一部分,在模擬胃、腸兩階段各別消化中,以含有胃蛋白酶之模擬胃消化液處理,結果顯示 FIP-fve 並不會有所降解,而在含有胰酶之模擬腸道消化液中,FIP-fve 雖少部分被消化並產生兩個片段,但大部分並未被消化。使用模擬連續式腸胃道消化 FIP-fve,其經模擬胃消化兩小時後,再進行模擬腸消化兩小時,透過高效能液相層析儀分析結果顯示 FIP-fve 並無降解。第二部分,實驗發現 FIP-fve 可以刺激腸繫膜淋巴結 (mesenteric lymph node, MLN) 及培耶氏斑 (Peyer’s patches, PP) 細胞產生 IFN-γ,此外,亦發現消化後的 FIP-fve 仍具有調節腸道免疫之活性,可刺激培耶氏斑細胞分泌 IFN-γ,因此推論 FIP-fve 之口服穩定性佳,模擬腸胃道消化並不會使 FIP-fve 降解,可通過消化道到達小腸,具良好之生物可及性。且經消化之 FIP-fve尚有調節腸道免疫之功能,可能進而影響全身性的免疫反應,達到治療疾病的效果。

FIP-fve is an immunomodulatory protein which is purified from Flammulina velutipes. It has been investigated that FIP-fve has the abilities to stimulate the proliferation and the production of cytokine IFN-γ of human peripheral blood mononuclear cells (hPBMC) and skew the T cells toward TH1 immune response. Besides, in vivo studies indicated that the effects of orally administrated FIP-fve in decreasing food-allergic reaction in mice, inhibiting the progression of tumor size and prolonging the life of mice bearing liver cancer. Bioaccessibility stands for the availabilities of bioactive compounds released from food matrix after passing through gastrointestinal tract which is determined by mimicking digestive system in vitro. In this study, we would like to investigate the stability of immunomodulatory protein FIP-fve after passing through gastrointestinal digestion and determine whether its digested product could further activate the cells of gut-associated lymphoid tissue (GALT).
Results showed that during mimicking each stage of digestion in stomach and intestine, FIP-fve was not degraded under the presence of digestive enzyme which contained pepsin. Moreover, after incubated in simulated intestinal fluid (SIF) which contained pancreatin, FIP-fve was slightly digested into fragments but not digested completely. In addition, the mimicked continuous gastrointestinal digestion of FIP-fve was performed for 2 hours. In this case, results determined via HPLC showed that FIP-fve was not degraded during the mimicked gastrointestinal digestion. Furthermore, digested FIP-fve could trigger the activation of mesenteric lymph node (MLN) and Peyer’s patch (PP) cells to generate IFN-γ. Results reveals the effects that the modulation of immune responses on GALT was achieved by stimulating IFN-γ production from Peyer’s patch.
Briefly, we could conclude that orally intake of FIP-fve is considerably stable to reach intestine against gastrointestinal digestion. In addition, the potential of immunomodulatory effects of FIP-fve may further affect systemic immune responses and has the possibility to be utilized on therapy studies.

口試委員會審定書 I
摘要 II
ABSTRACT III
目錄 V
表目錄 VII
圖目錄 VIII
第一章 前言 1
第一節 金針菇簡介 1
第二節 生物可及性 (bioaccessibility) 5
第三節 腸道黏膜免疫系統簡介 7
第四節 研究動機與實驗架構 10
第二章 材料與方法 11
第一節 免疫調節蛋白 FIP-fve 之純化 11
第二節 SDS-PAGE 14
第三節 小鼠脾細胞取得 16
第四節 體外模擬胃消化 17
第五節 體外模擬腸道消化 18
第六節 體外模擬胃腸連續消化 19
第七節 以高效液相層析儀 (HPLC) 分析消化後產物 21
第八節 分離腸繫膜淋巴結細胞 22
第九節 分離腸道 Peyer’s patches 細胞 23
第十節 流式細胞儀分析細胞表面分子 25
第十一節 CFSE 標定細胞增生試驗 26
第十二節 酵素連結免疫吸附分析細胞激素 28
第十三節 統計方法 29
第三章 結果 30
第一節 FIP-fve 之純化及其刺激脾細胞之增生並產生 IFN-γ 30
第二節 體外模擬胃腸消化 FIP-fve 31
第三節 體外模擬腸胃道連續消化 FIP-fve 33
第四節 分離 BALB/c 小鼠之 Peyer’s patches 及 MLN並分析其細胞分群 34
第五節 模擬腸胃道消化後之 FIP-fve 刺激 Peyer’s patches 細胞之活性 36
第六節 經模擬腸胃道消化後之 FIP-fve 刺激 MLN 分泌 IFN-γ 37
第四章 討論 39
第一節 FIP-fve 之免疫調節活性 39
第二節 FIP-fve 之消化穩定性 40
第三節 FIP-fve 具藉由調節腸道免疫影響系統性免疫之潛力 41
第四節 結論及未來展望 42
第五章 參考文獻 44
TABLES 50
FIGURES 51
附錄 67

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