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研究生:楊世昂
研究生(外文):Shih-Ang Yang
論文名稱:香菇菌絲多醣體活化小鼠巨噬細胞株RAW264.7之探討
論文名稱(外文):Macrophage RAW264.7 Activation by Polysaccharide from Lentinus edodes mycelia
指導教授:吳美莉吳美莉引用關係
指導教授(外文):Mei-Li Wu
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:73
中文關鍵詞:香菇菌絲體一氧化氮合成酶NFκB巨噬細胞
外文關鍵詞:Lentinus edodes myceliaiNOSNFκBMacrophage
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巨噬細胞是具有高吞噬性功能的細胞,全身廣泛散佈,在宿主體內扮演著調控先天性免疫和發炎反應的重要角色。多醣體β-glucan為菇蕈類細胞壁上的主要成分之ㄧ,可活化體內多種免疫細胞、提高巨噬細胞吞噬能力,並促進細胞激素與干擾素的生成。本研究以體外試驗進行香菇菌絲多醣體對巨噬細胞活化及生理活性之探討。我們發現香菇菌絲多醣體刺激細胞增生測試中,發現隨著濃度作用增加,對細胞有增生之趨勢,且有增加一氧化氮產生量的效果,並顯著提升巨噬細胞吞噬能力及胞內ROS之提升。經由西方墨點法分析蛋白表現,iNOS表現量隨著香菇菌絲體作用濃度提高而上升,更進一步探討胞內轉錄蛋白機制,發現隨著香菇菌絲多醣體作用時間增加,IκB蛋白有磷酸化之現象,因而活化轉錄因子NFκB p65移轉至細胞核內。結果顯示香菇菌絲多醣體之活化巨噬細胞與NFκB路徑誘發iNOS之路經有關。
Macrophages are phagocytic cells widely distributed throughout the body, and play essential role in innate immunity and inflammatory responses. Polysaccharides β-glucan is the major component of mushroom cell walls which can activate immuno cells, increased phagocytic activity of macrophage, cytokine and interferon production. In this study, we attempt to investigate the biological activity of polysaccharide of Lentinus edodes mycelia (LEM) on activation of macrophage in vitro. We found that polysaccharide of LEM strongly upregulate the phenotypic functions of macrophages such as phagocytic uptake, ROS/NO production and morphological changes. Western immunoblotting assays revealed that polysaccharide of LEM induced NO production of RAW264.7 macrophage through the increased expression of inducible nitric oxide synthase (iNOS), stimulated phosphorylation and degradation of IκB-α and subsequent NFκB p65 activation. Therefore, our data suggest that polysaccharide of LEM may promote macrophage activation via NFκB signaling pathways.
摘要 I
Abstract II
謝誌 IV
目錄 V
圖表目錄 VIII
第1章 前言 1
第2章 文獻回顧 4
2.1 香菇菌絲體(Lentinus edodes mycelia) 4
2.2 菇類之免疫生理活性 4
2.3 免疫反應 6
2.4 巨噬細胞(Macrophage) 6
2.5 多醣體β-glucan特性 7
2.6 多醣結構及活性之間的關係 9
2.7 β-glucan與免疫細胞的關係 10
2.8 一氧化氮(Nitric Oxide, NO) 11
2.9 一氧化氮的免疫反應 13
2.10 活性氧物質(Reactive oxygen species, ROS) 13
2.11 NFκB 15
2.12 一氧化氮合成酶(nitric oxide synthase, NOS)的表現 15
第3章 材料與方法 18
3.1 研究目的(Aim of experiment) 18
3.2 實驗架構 19
3.3 細胞株 (Cell line) 20
3.4 儀器設備 20
3.5 藥品試劑 20
3.5.1 樣品 20
3.5.2 抗體 21
3.5.3 常用藥品 21
3.6 樣品製備及初步結構分析 22
3.6.1樣品製備來源 22
3.6.2 多醣總醣含量測定 22
3.6.3 傅力葉紅外線光譜分析 23
3.7 細胞解凍 23
3.8 細胞繼代培養 23
3.9 細胞計數 23
3.10 細胞存活率分析(MTT assay) 23
3.11 Nitrite 測定與分析 24
3.12 蛋白質電泳與西方墨點法 25
3.12.1 蛋白質萃取 25
3.12.2 蛋白質定量 26
3.12.3 蛋白質電泳分析 27
3.12.4 轉印與免疫染色法 29
3.13 細胞內ROS產量之分析 31
3.14 老鼠巨噬細胞株RAW264.7吞噬作用之影響 31
3.15 老鼠巨噬細胞株RAW264.7吞噬螢光E.coli之觀察 32
3.16 免疫螢光分析(Immunofluorescence) 32
第4章 結果 33
4.1 香菇菌絲多醣體總醣量分析 33
4.2 傅力葉紅外線光譜(FTIR)分析 33
4.3 細胞存活率偵測分析(MTT assay) 33
4.4 巨噬細胞型態活化 34
4.5 Nitrite的測定與分析 34
4.6 香菇菌絲多醣體刺激RAW264.7吞噬作用之影響 35
4.7 ROS之測定 35
4.8 iNOS 酵素蛋白分析 36
4.9 香菇菌絲多醣體刺激RAW264.7細胞轉錄因子NFκB轉位偵測 37
4.10 IκBα蛋白質量分析 37
第5章 討論 55
第6章 結論 58
參考文獻 60
作者簡介 73

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