樟芝 (Antrodia cinnamomea) 為台灣真菌特有種，為具有保健療效潛力之藥用真菌。本研究室之前已自樟芝菌絲體發酵液純化出免疫調節蛋白 ACA1 並選殖出其核酸序列，且於大腸桿菌中進行異體表現，以大量生產重組 ACA1 蛋白。本研究即以重組 ACA1 之融合蛋白 (frACA1) 為試驗樣品，希望進一步瞭解樟芝免疫調節蛋白對於活體巨噬細胞的活化作用。利用 15 μg/mL frACA1 處理小鼠腹腔巨噬細胞 (peritoneal macrophage，pMΦ)，可增進 pMΦ

Antrodia cinnamomea is not only an endemic fungus but also a potential medicinal mushroom in Taiwan. Our laboratory had previously isolated an protein, named ACA1 and demonstrated its immunomodulatory ability to activate murine macrophages, from the mycelia of A. cinnamomea. The gene encoding ACA1 had also been cloned and expressed in E.coli for the production of recombinant ACA1 protein. In this study, the macrophage-stimulating activity of recombinant ACA1 fusion protein (frACA1) was thoroughly studied. The results showed that treatment with 15 μg/mL frACA1 increased the phagocytic ability and CD86/B7-2 expression of mouse peritoneal macrophages (pMΦ). In addition, real-time PCR results demonstrated that frACA1 could up-regulate the mRNA expression of cytokines, including IL-1β, TNF-α, IL-6, and iNOS, in a time dependent manner, which suggested that frACA1 could activate pMΦ without hyperinflammation. Moreover, frACA1 promoted the mRNA expression of IL-12p35 and M1 type chemokines genes, including CCL3, CCL4, CCL5 CXCL10, in murine pMΦ. These results revealed that frACA1 was capable to promote Th1 immune response by inducing M1 polarization of pMΦ. Furthermore, we investigated whether frACA1-induced pMΦ activation was mediated with TLR2- or TLR4-signaling pathway or not. The results showed that frACA1 could induce TNF-α secretion by the pMΦ of both TLR2 and TLR4 deficiency mouse. It was also observed that frACA1 showed insignificant effect on increasing the mRNA expression of TLR2, TLR4, MyD88, and NF-κB gene in BALB/c mouse pMΦ (p>0.05), suggesting that TLR2- and TLR4-signaling pathway were not involved in frACA1-stimulating pMΦ activation and the possibility of LPS-contamination in frACA1 was also excluded. However, flowcytometry indicated that there was no interaction between frACA1 and cell surface of pMΦ. Therefore, further investigation could be carried out to discover the activation pathway of frACA1 on murine pMΦ.

目錄 I
圖表目錄 IV
中文摘要 1
英文摘要 3
第一章 前人研究 5
第一節 前言 5
第二節 食藥用菇菌類的重要性 5
第三節 樟芝與其相關研究之介紹 8
第四節 樟芝免疫調節蛋白之發現與研究 15
第五節 樟芝免疫調節蛋白對巨噬細胞可能的調節作用 19
第六節 研究動機與構想 31
第二章 材料與方法 34
第一節 樟芝免疫調節蛋白之製備 34
第二節 小鼠腹腔巨噬細胞之取得 45
第三節 小鼠腹腔巨噬細胞吞噬活性測試 48
第四節 小鼠腹腔巨噬細胞抗原呈獻能力測試 50
第五節 細胞激素 ELISA 測定法 51
第六節 一氧化氮測定法 53
第七節 腹腔巨噬細胞基因表現量之測定 54
第八節 TLRs 與 frACA1 活化巨噬細胞之相關性 60
第九節 frACA1 與小鼠腹腔巨噬細胞之親和作用 61
第十節 統計分析方法 63
第三章 結果 64
第一節 重組樟芝免疫調節蛋白 frACA1 之製備與活性確認 64
第二節 frACA1 對巨噬細胞吞噬能力的影響 67
第三節 frACA1 對巨噬細胞抗原呈獻能力的影響 68
第四節 frACA1 可增加細胞激素之 mRNA 表現與分泌 70
第五節 frACA1 對巨噬細胞極化之影響 72
第六節 TLRs 路徑與 frACA1 活化巨噬細胞之相關性 75
第七節 frACA1 與巨噬細胞表面之親和作用 77
第四章 討論 79
第一節 frACA1 樣品之選擇 79
第二節 frACA1 對巨噬細胞吞噬及呈獻抗原功能的影響 80
第三節 frACA1 可增進細胞激素之 mRNA 表現與分泌 81
第四節 frACA1 與 IFN-γ 共同作用可明顯增加 NO 分泌量 82
第五節 frACA1 對於巨噬細胞極化之影響 83
第六節 TLRs 路徑與 frACA1 活化巨噬細胞之相關性 85
第七節 frACA1 與小鼠腹腔巨噬細胞表面之親和作用 88
第八節 frACA1 活化作用的可能機制 90
第五章 結論 92
參考文獻 93
圖表 100
附件 122


圖表目錄
表一、frACA1所含內毒素之檢測結果 100
表二、frACA1 對巨噬細胞極化作用的影響 101
圖一、研究架構 102
圖二、frACA1 之製備 103
圖三、frACA1 與 rACA1 對 BALB/c 小鼠腹腔巨噬細胞 TNF- 與 NO 分泌量之影響 104
圖四、frACA1 對 BALB/c 小鼠腹腔巨噬細胞吞噬活性之影響 105
圖五、frACA1 對 BALB/c 小鼠腹腔巨噬細胞表現 CD80 之影響 106
圖六、frACA1 對 BALB/c 小鼠腹腔巨噬細胞表現 CD86 之影響 107
圖七、frACA1 對 BALB/c 小鼠腹腔巨噬細胞表現 MHC class II 之影響 108
圖八、frACA1 對 BALB/c 小鼠腹腔巨噬細胞細胞激素與 iNOS mRNA 表現量之影響 109
圖九、frACA1 對 BALB/c 小鼠腹腔巨噬細胞 TNF-α、IL-1β、NO 分泌量之影響 110
圖十、IFN-γ對 frACA1 活化小鼠腹腔巨噬細胞分泌 TNF-α與 NO 之影響 111
圖十一、frACA1 對 BALB/c 小鼠腹腔巨噬細胞 M1 型趨化素 mRNA 表現量之影響 112
圖十二、frACA1 對 BALB/c 小鼠腹腔巨噬細胞 M2 型趨化素 mRNA 表現量之影響 113
圖十三、frACA1 對小鼠腹腔巨噬細胞 IL-10 與 IL-12p35 mRNA 表現量之影響 114
圖十四、frACA1 對 TLR2-/- 免疫缺陷小鼠腹腔巨噬細胞分泌TNF-α 效應之影響 115
圖十五、frACA1 對 TLR4-/- 免疫缺陷小鼠腹腔巨噬細胞分泌TNF-α 效應之影響 116
圖十六、frACA1 對於 TLR4-/- 免疫缺陷小鼠腹腔巨噬細胞之細胞激素及趨化素 mRNA 表現的影響 117
圖十七、frACA1 對於小鼠腹腔巨噬細胞之 TLR2 與 TLR4 受器 mRNA 表現之影響 118
圖十八、frACA1 對於小鼠腹腔巨噬細胞之 TLRs 路徑下游訊息傳導分子 mRNA 表現之影響 119
圖十九、以 SDS-PAGE 與 Syngene 螢光偵測系統分析 FITC 螢光標定frACA1 120
圖二十、以流式細胞儀分析 frACA1 與小鼠腹腔巨噬細胞表面之交互作用 121
附件一、pET30Aca 載體之圖譜 122
附件二、本研究中所使用的 real-time PCR 引子對 123

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