樟芝（Antrodia cinnamomea）是台灣發現的特有物種，是一種廣泛應用於傳統中藥上的真菌，相傳具有抗腫瘤、抗氧化及抗過敏等功效。樟芝已被鑑定出含有多種生理活性成分，包括：多醣體（polysaccharides）、三帖類（triterpenoids）、超氧歧化酵素（superoxidase dismutase, SOD）、腺苷酸（adenosine）、蟲草素 （cordycepin）及麥角固醇（ergosterol）等。本研究探討以固態培養樟芝甲醇萃取物 （Antrodia cinnamomea extracts, ACE）及其生理活性成分－腺苷酸（adenosine）、蟲草素（cordycepin）與麥角固醇（ergosterol）對於脂多醣（Lipopolysaccharide, LPS）所誘發巨噬細胞（RAW 264.7）發炎反應之影響。結果顯示，ACE 及其生理活性成分－ adenosine 、 cordycepin 及 ergosterol 可抑制 LPS （1 μg/mL）誘發巨噬細胞所產生之 TNF-α 含量，在蛋白質分析結果亦有效抑制 iNOS（inducibie nitric oxide synthase）及 NF-kB 表現。此外，利用 ACE 及其生理活性成分作用於 RAW 264.7 探討其抑制細胞存活率及可能作用機制。利用細胞毒性試驗、流式細胞儀Annexin V-FITC/PI 雙染法並以顯微鏡觀察細胞的型態變化來觀察細胞凋亡情形。結果顯示，ACE 及其生理活性成分均造成巨噬細胞 RAW 264.7 存活率（viability）下降，但僅有 ACE 及 cordycepin 能夠誘導細胞凋亡。本研究並以蛋白質體分析系統研究細胞內蛋白質表現變化，結果共有 32 個表現量差異性在 1.5 倍以上之蛋白質量，經由質譜儀 （MALDI-Q-TOF）被鑑定出與資料庫比對及蛋白質功能鑑定分析；結果獲得與發炎反應與細胞凋亡有關的蛋白質，包括 RhoGDI、Arrβ1、Eras 皆能藉抑制 GTP 結合蛋白而抑制細胞內訊息傳導，並藉由抑制 TCPθ 蛋白使其後續骨架蛋白之表現受抑制；此外，具有活化 MAPK 路徑相關的 Ep11蛋白質表現可能與細胞凋亡的發生有關。在抑制發炎反應方面能夠直接抑制 TANK 蛋白表現而影響 NF-κB 活化穩定，同時也可藉由調控 HSP-90 及 TR163蛋白質進而影響細胞內 proteosome 將 IκB kinase 水解進而抑制轉錄因子 NF-κB 與其後續發炎蛋白質表現。

Antrodia cinnamomea (formerly Antrodia camphorata) is a well-known medicinal fungus used to treat inflammation and cancer in Taiwan. Though several bioactive components from A. cinnamomea have been identified, including adenosine, cordycepin, ergosterol triterpenoids, and polyphenols, very little is currently known about the anti-inflammatory mechanisms underlying the independent or synergetic action of the single bioactive component. In this study, we investigated anti-inflammatory mechanisms effects of A. cinnamomea extract (ACE), adenosine, cordycepin and ergosterol. In previous studies, we have shown that ACE (200 μg/mL), adenosine (100 μg/mL), cordycepin (50 μg/mL) and ergosterol (10 μg/mL) not only successfully inhibited Lipopolysaccharide (LPS, 1 μL/mL)-induced inflam- matory responses, but also decreased nitrite and TNF-α production in RAW 264.7 macrophage cells. Furthermore, ACE, adenosine and cordycepin inhibited iNOS (inducibie nitric oxide synthase), COX-2 (coclooxygenase-2) protein expression by Wentern blot analysis. Next, flow cytometry analysis with PI and Annexin V-FITC was used to examine the relative amount of apoptosis induction for ACE, adenosine, cordycepin and ergosterol. we have shown that ACE and cordycepin induced a dose-dependent increase in the number of apoptotic cells, but was not observed by adenosine and ergosterol treatment.
In this study, a proteomic approach was used to compare the modify- cations in protein expression in LPS-activated RAW 264.7 macrophage cells treated with ACE, adenosine, cordycepin and ergosterol. When comparing the proteomes, multiple protein alterations involving NF-κB cascade-related proteins and chaperones were observed, and validated by western blotting. These results indicated may exert an anti-inflammatory action via heat shock protein 90 (HSP90) inhibition- mediated suppression in the proteasome activity to inactivate the NF-κB signaling pathway.

目錄
目錄 Ⅰ
圖目錄 IV
表目錄 VI
附錄目錄 VII
中文摘要 VIII
英文摘要 Ⅸ
第一章 前 言 1
第二章 文獻回顧 2
一、樟芝的簡介 2
二、樟芝之生理活性成分 3
（一）多醣體 4
（二）三萜類 4
（三）腺苷酸 4
（四）蟲草素 5
（五）麥角固醇 6
三、巨噬細胞及其功能 7
四、脂多醣活化巨噬細胞之機制 8
五、發炎反應與巨噬細胞之關連 8
（一）NF-κB轉錄因子之簡介 8
（二）NO與iNOS與疾病之關連性 9
六、細胞凋亡及其路徑 10
七、蛋白質二維電泳 12
八、研究動機與論文架構 13
第三章 材料與方法 15
第一節 實驗材料 15
（一）樣品來源 15
（二）細胞株 15
（三）實驗藥品與材料 15
（四）套裝試劑組與抗體 18
（五）實驗器材與設備 19
第二節 實驗方法 21
（一）樣品之製備 21
（二）細胞株培養 22
（三）細胞繼代培養 22
（四）細胞保存與活化 23
（五）細胞處理模式 23
（六）細胞內毒性試驗 24
（七）原漿微粒蛋白質之分離 24
（八）蛋白質濃度分析 25
（九）SDS-PAGE蛋白質膠體電泳 26
（十）西方點墨法 26
（十一）使用流式細胞儀觀察細胞凋亡 28
（十二）細胞激素TNF-α分泌之測定 29
（十三）細胞分泌一氧化氮之測定 30
第三節 二維蛋白質膠體電泳分析實驗 30
（一）二維白質膠體電泳細胞蛋白質樣品製備 30
（二）二維電泳之蛋白質萃取液定量 31
（三）一維蛋白質膠體電泳樣品前處理 31
（四）第一維蛋白質膠體電泳 32
（五）SDS電泳膠片的製作 32
（六）二維蛋白質膠體電泳 33
（七）二維電泳膠片染色 33
（八）二維電泳膠片影像分析 34
（九）膠體內蛋白質分解 35
（十）蛋白質身分鑑定 36
第四章 實驗結果 37
一、顯微觀察細胞之型態變化 37
二、樟芝萃取物對於巨噬細胞RAW 264.7毒殺作用之結果 40
三、樟芝萃取物對於巨噬細胞RAW 264.7分泌NO與細胞激素TNF-α 產生之結果 42
四、流式細胞儀分析樟芝萃取物對於巨噬細胞RAW 264.7凋亡之結果 43
五、使用蛋白質體學分析樟芝萃取物處理巨噬細胞 RAW 264.7之結果 46
六、樟芝萃取物對於巨噬細胞RAW 264.7之 iNOS及COX-2 蛋白質表現之結果 51
七、樟芝萃取物對於巨噬細胞RAW 264.7之NF-κB、p-IκB-α 及 HSP90蛋白質表現之結果 52
第五章 討 論 54
第六章 結 論 59
附 錄 102
參考文獻 109
縮寫表 117



圖目錄
圖一、腺苷酸、蟲草素及麥角固醇之結構式 7
圖二、細胞程序性凋亡及壞死之差異性 11
圖三、使用顯微鏡觀察巨噬細胞RAW 264.7 經LPS刺激24小時細胞形態之變化 60
圖四、使用顯微鏡觀察不同濃度ACE處理 LPS刺激之RAW 264.7巨噬細胞 12小時及24小時之細胞形態 61
圖五、使用顯微鏡觀察不同濃度腺苷酸處理 LPS刺激之RAW 264.7巨噬細胞 12小時及 24小時之細胞形態 62
圖六、使用顯微鏡觀察不同濃度蟲草素處理 LPS刺激之RAW 264.7巨噬細胞 12小時及 24小時之細胞形態 63
圖七、使用顯微鏡觀察不同濃度麥角固醇處理 LPS刺激之RAW 264.7巨噬細胞 12小時及 24小時之細胞形態 64
圖八、使用MTT 試驗分析樟芝萃取物處理經LPS刺激之RAW 264.7巨噬細胞株24小時之細胞抑制率 65
圖九、使用 MTT 試驗分析腺苷酸處理經LPS刺激之 RAW 264.7巨噬細胞株24小時之細胞抑制率 66
圖十、使用 MTT 試驗分析蟲草素處理經LPS刺激之 RAW 264.7巨噬細胞株24小時，之細胞抑制率 67
圖十一、使用 MTT 試驗分析麥角固醇處理經LPS刺激之 RAW 264.7巨噬細胞株24小時之細胞抑制率 68
圖十二、不同濃度之樟芝萃取物、腺苷酸、蟲草素及麥角固醇處理LPS刺激之 RAW 264.7巨噬細胞後所產生TNF-α 含量之結果 69
圖十三、不同濃度之樟芝萃取物、腺苷酸、蟲草素及麥角固醇處理LPS刺激之 RAW 264.7巨噬細胞株後培養液中 NO 含量之結果 70
圖十四、使用 Annexin V-FITC/PI雙染法偵測巨噬細胞RAW 264.7對於不同濃度之樟芝萃取物處理12小時之變化 71
圖十五、使用 Annexin V-FITC/PI雙染法偵測巨噬細胞 RAW 264.7對於不同濃度之樟芝萃取物處理24小時之變化 72
圖十六、使用 Annexin V-FITC/PI雙染法偵測巨噬細胞 RAW 264.7對於不同濃度之腺苷酸處理24小時之變化 73
圖十七、使用 Annexin V-FITC/PI雙染法偵測 RAW 264.7細胞對於不同濃度之蟲草素處理24小時之變化 74
圖十八、使用 Annexin V-FITC/PI雙染法偵測巨噬細胞 RAW 264.7對於不同濃度之麥角固醇處理24小時之變化 75
圖十九、使用流式細胞儀測定分樟芝萃取物、腺苷酸、蟲草素及麥角固醇對於 RAW 264.7巨噬細胞株凋亡率之變化 76
圖二十、樟芝萃取物處理 RAW 264.7巨噬細胞之二維電泳圖 77
圖二十一、腺苷酸萃取物處理 RAW 264.7巨噬細胞之二維電泳圖 80
圖二十二、蟲草素處理 RAW 264.7巨噬細胞之二維電泳圖 81
圖二十三、麥角固醇處理 RAW 264.7巨噬細胞之二維電泳圖 82
圖二十四、西方點墨法分析樟芝萃取物、腺苷酸、蟲草素及麥角固醇對於RAW 264.7巨噬細胞株之iNOS及 COX-2蛋白表現之影響 100
圖二十五、西方點墨法分析樟芝萃取物、腺苷酸、蟲草素及麥角固醇對於RAW 264.7巨噬細胞株之NF-κB、p-IκB-α 及 HSP-90蛋白表現之影響 101

表目錄
表一、使用二維電泳分析經樟芝萃取物、腺苷酸、蟲草素及麥角固醇處理RAW 264.7細胞所誘導蛋白質變化之結果 87
表二、樟芝萃取物、腺苷酸、蟲草素及麥角固醇處理後所誘導變化之蛋白質功能性及生物程序 91

附錄目錄
附錄一、樟芝樣品腺苷酸、蟲草素及麥角固醇成分分析結果 105
附錄二、三萜類HPLC層析圖 105
附錄三、老鼠 TNF-α 之標準曲線 106
附錄四、亞硝酸鈉（NaNO2）以Griess reaction 測定之標準曲線 107
附錄五、資料庫之搜尋條件（A）PMF（B）MS/MS 108

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