臺灣博碩士論文加值系統

姬松茸蘑菇(Agaricus blazei Murill)原產於巴西，又名為巴西蘑菇，是一種食藥用菇，近年來被廣泛地進行研究，而其 β-D-葡聚糖含量特別高，為菇類之冠，具有獨特的抗癌功效兼具抗腫瘤效果。本研究以姬松茸多醣體連續餵食 BALB/c 小鼠不同時間點，觀察其是否具有抗發炎及增強免疫力之功效。研究內容分析小鼠餵食姬松茸多醣體對其脾臟免疫細胞，如巨噬細胞、T 細胞、樹突狀細胞所產生之作用。研究結果顯示，長期餵食小鼠姬松茸多醣體，再給予腹腔注射 LPS 使其產生發炎反應，經流式細胞儀測定分析，發現小鼠脾臟上巨噬細胞之 Trem-1HI 的表現受到抑制而下降；樹突狀細胞上 CD86HI 表現亦顯示下降的趨勢；樹突狀細胞上之 CD40HI、CD8αHI 的表現則均增加。另外 T 細胞上 ICOS 的表現量則於長期餵食姬松茸多醣體後增加，此結果與抗原呈獻細胞上 CD86 的表現下降相反，其原因可能是 CTLA-4 參與調控平衡而抑制 CD86 的表現。此外，利用 real-time PCR 觀察促發炎因子，顯示相關發炎之細胞激素如 Trem-1、TNF-α、IFN-γ、iNOS、IL-1β、IL-6、IL-12、CCL2 及 PGE2 均有被抑制的現象，發現長期餵食姬松茸多醣體確實具有抗發炎的效果。
本論文研究結果顯示餵食小鼠姬松茸多醣體後，具有提升免疫力及抗發炎之功效，並從中探討各種免疫分子間相關作用機轉。本研究中所使用的細胞分子表面標記可考慮作為藥物免疫作用篩選的標的物，用來測試其他具有潛力的中草藥或健康食品。

Agaricusl blazei Murill has been traditionally used as a health food source in Brazil for the prevention of cancer, diabetes, hyperlipidaemia, arteriosclerosis and chronic hepatitis. In this study, BALB/c mice were fed with A. blazei polysaccharides at different time points to investigate the anti-inflammatory action and the immunomodulatory effect. When mice were administrated A. blazei polysaccharides for a period of time and then injected LPS into mice, The Trem-1HI expression on macrophages is inhibited. The expressions of CD40HI and CD8αHI markers on DCs increased apparently. However CD8HI maker on macrophages decreased significantly, this result may regulated by the activation of CTL-4 and ICOS from T cells. In addition, we use real-time PCR to detect Trem-1、TNF-α、IFN-γ、iNOS、IL-1β、IL-6、IL-12、CCL2 and PGE2 mRNA cytokine inhibited obviously. The results mice demonstrated A. blazei polysaccharides possesses effects on both immunomodulation and anti-inflammation. These makers may serve as targets for further understanding the molecular basis and efficacy of A. blazei polysaccharides and other Chinese herbal medicines in the future.

中文摘要 I
ABSTRACT II
目 錄 III
圖 目 錄 VII
表 目 錄 X
第一章 緒論 1
一、 研究動機 1
二、 研究目的 1
三、 生物體之免疫調節機制 2
四、 發炎作用指標 5
第二章 文獻回顧 11
一、 大型真菌類之生理活性 11
二、 姬松茸的介紹 13
(一) 姬松茸之發現與由來 13
(二) 姬松茸的型態特徵及分類 15
(三) 姬松茸之化學組成 15
(四) 姬松茸的生理活性 18
(五) 姬松茸的安全性 19
三、菇類活性成份探討 20
(一)菇類多醣體的介紹 20
(二) 姬松茸之多醣體 22_Toc333838948
(三) 姬松茸之麥角固醇 25
第三章 材料與方法 27
一、 實驗材料 27
(一) 姬松茸多醣體之製備 27
(二) 實驗動物 27
二、 實驗設計 27
三 實驗方法 29
(一)小鼠脾臟細胞之取得 30
(二) 以流式細胞技術分析偵測小鼠脾臟免疫細胞表面標幟 31
1、以流式細胞儀偵測 31
(三) 統計分析方法 32
(四) 以即時定量反轉錄聚合酶連鎖反應（Real-Time polymerase chain reaction）分析發炎相關細胞激素之mRNA表現 34
1、純化小鼠脾臟細胞之mRNA 34
2、反轉錄步驟－樣品去chromosome DNA後及互補去氧核糖核酸（cDNA）之合成 34
3、即時定量反轉錄聚合酶連鎖反應（Real-Time polymerase chain reaction） 35
(五) Real-Time PCR反應條件設計為 35
第四章 結果 37
一、姬松茸多醣體對小鼠脾臟免疫細胞之調控 37
(一) 免疫細胞表面標記測定 37
(二) 小鼠脾臟-DC 之表現 37
1、DC 的細胞數量(CD11c+)百分比 37
2、CD40HI 在 DC 之表現 39
3、CD8αHI 在 DC 之表現 41
4、CD86HI 在 DC 之表現 43
(三)小鼠脾臟細胞-Macrophage 之表現 45
1、Macrophage 的細胞數量(CD11b+)百分比 45
2、CD8αHI 在 Macrophage 之表現 47
(四) 小鼠脾臟-T 細胞之表現 49
1、CD4+ T cell之表現 49
2、CD8α+ T cell 之表現 51
3、ICOSHI 在 CD4+ T cell之表現 53
(五)姬松茸抗發炎功效之細胞診斷 55
1、Macrophage 表面發炎指標性Marker（Trem-1HI）測定 55
二、以Real-time PCR分析 Trem-1 以及其它發炎相關細胞激素之mRNA表現 57
(一) Trem-1 mRNA 之表現 57
(二) TNF-α mRNA 之表現 59
(三) iNOS mRNA 之表現 59
(四) IFN-γ mRNA 之表現 60
(五) IL-1β mRNA 之表現 62
(六) IL-6 mRNA 之表現 62
(七) IL-12 mRNA 之表現 64
(八) CCL2 mRNA 之表現 64
第五章 討論 68
參考文獻 74
附錄一：縮寫對照表 81

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