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研究生:張耕銘
研究生(外文):Geng-Ming Jhang
論文名稱:牛樟芝萃取物對牛隻的免疫調控評估
論文名稱(外文):Evaluation of the immunomodulatory effects of Antrodia camphorata extract on dairy cows
指導教授:李嘉偉李嘉偉引用關係朱純燕
指導教授(外文):Jai-Wei LeeChun-Yen Chu
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:動物疫苗科技研究所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:82
中文關鍵詞:牛樟芝佐劑多醣體免疫調控
外文關鍵詞:Antrodia camphorataajuvantpolysaccharidesimmunomodulatory
相關次數:
  • 被引用被引用:1
  • 點閱點閱:1979
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  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:1
牛樟芝(Antrodia camphorate;AC)屬於多孔菌科,是一種台灣所特有且珍貴的菌種,生長於牛樟樹(Cinnamomum kanehirai)的內壁上。從先前的研究結果,我們得知牛樟芝處理後所得到的萃取物,具有抗氧化和抗過敏以及對抗肝炎還有癌症的效用,以及對免疫系統的調控。而在之前文獻中的動物試驗,所選用的模式動物皆為小鼠,並無大型動物的試驗,因此我們將選用牛隻為模型進行牛樟芝萃取物對大動物免疫調控的觀察。結果顯示,從牛血中分離出的周邊血液單核球細胞(Peripheral Blood Mononuclear Cell;PBMC) 所進行的體外試驗,在濃度為50 μg /mL時,能夠有效地刺激細胞的生長,但是當多醣體過量時會出現抑制作用(>100 μg/mL)。動物試驗中,搭配標準抗原KLH (Hemocyanin From Keyhole Limpets)施予低濃度之多醣(每劑含100 μg),能夠提升牛隻體內的抗體力價,並且引發顯著的IgG2抗體反應 (P<0.05)。在第一次免疫後四週,T淋巴球細胞能有效分化成為CD8+ T細胞。綜合試驗結果,樟芝萃取物應用於牛隻體內,是具有免疫促進之功效,但最佳的免疫濃度需再進一步釐清。
Antrodia camphorata (AC) is a rare and indigenous mushroom of the family Polyporaceae that only grows in the inner cavity of Cinnamomum kanehirai Hay. Previously studies demonstrated that AC extracts have anti-oxidant, anti-inflammatory, anti-hepatitis, anti-tumor, and immunomodulatory effects. However, most of the studies related to AC were carried out in mice, but not other large animals. In the present study, dairy cows were used as the research model to examine the immunomodulatory effects of AC extract in vitro and in vivo. Results showed that the proliferation of bovine peripheral blood mononuclear cells (PBMC) was significantly increased by the stimulation of AC extract when the concentration was 50 μg /mL and overdoses (> 100 μg /mL) lead to negative effects. When AC extract was used as the adjuvant at two levels (100 and 500 μg) in a standard KLH (Hemocyanin From Keyhole Limpets) vaccine, cows immunized with the
vaccine containing 100 μg AC extract had significantly (P<0.05) increased KLH-specific antibody titer (IgG2) in serum and differentiation of CD8+ T cell at 4 weeks after the primary vaccination in comparison with other treatments. AC extract, when used as the adjuvant in a vaccine, can enhance Th1 immune responses. In summary, AC extract has immunostimulatory effects in the bovine model, however, the optimal concentration as adjuvant for bovine vaccines requires further investigations.

目 錄
摘要 I
Abstract II
謝誌 IV
目錄 V
圖表目錄 IX
第1章 緒言 1
第2章 文獻回顧 3
2.1佐劑 3
2.1.1 免疫調控 (Immunomodulation) 3
2.1.2 抗原呈獻作用 (Presentation) 4
2.1.3 免疫細胞對抗原的專一性提升 (Targeting) 4
2.1.4 促進毒殺型T淋巴球細胞(CTL)的免疫反應 4
2.1.5 抗原的堆積 (Depot generation) 5
2.1.6 傳統佐劑與新型佐劑 5
2.1.7 鋁膠佐劑 6
2.1.8 鋁膠佐劑之應用 6
2.2 牛樟芝簡介 7
2.2.1 牛樟芝的源由與命名 7
2.2.2 型態與構造 7
2.2.3 牛樟芝的培養 8
2.2.4 生物活性成分 8
2.2.4.1 萜類 (terpenoids) 8
2.2.4.2 多醣體 (polysaccharides) 9
2.2.5 牛樟芝的藥理活性 9
2.2.5.1抗氧化能力 10
2.2.5.2抗癌效用 10
2.2.5.3保護肝臟 10
2.2.5.4免疫調節作用 11
2.3 免疫系統 11
2.3.1 免疫細胞與反應 11
2.3.1.1 先天性免疫反應 12
2.3.1.2 後天性免疫反應 13
第3章 材料與方法 15
3.1 實驗材料 15
3.1.1 牛樟芝 15
3.1.2 試劑與藥品 15
3.1.3 溶液配製 16
3.2 實驗方法 16
3.2.1 牛樟芝多醣萃取 17
3.2.2 內毒素(Endotoxin)含量檢測 17
3.2.3 β-葡聚糖濃度測定 17
3.2.3.1 制定標準曲線 17
3.2.3.2 樣品處理 18
3.2.4 體外試驗 18
3.2.4.1 血液採集 18
3.2.4.2 周邊血液單核球細胞(PBMC)及血清分離 18
3.2.4.3 淋巴球增生試驗 19
3.2.4.4細胞激素之基因表現量測定 19
3.2.4.4.1 細胞分組及處理 20
3.2.4.4.2 RNA萃取 20
3.2.4.4.3 RT-PCR 20
3.2.4.4.4 Real-time PCR 20
3.2.5 動物試驗 21
3.2.5.1 實驗動物分組 21
3.2.5.2 疫苗製備 22
3.2.5.3 樣品前處理 22
3.2.5.4 淋巴球細胞增生試驗 22
3.2.5.5 細胞激素之基因表現量測定 22
3.2.5.6 CD4+/CD8+ T 淋巴球 23
3.2.5.7 酵素連結免疫吸附分析法(Enzyme linked immunesorsorbent assay) 23
3.2.6 統計分析 23
第4章 結果 25
4.1 體外試驗 25
4.1.1內毒素(Endotoxin)含量檢測 25
4.1.2 牛樟芝多醣萃取液定量 25
4.1.3 淋巴球增生試驗 25
4.1.4 細胞激素之基因表現量 26
4.2 動物試驗 26
4.2.1細胞表面標記 CD4+ / CD8+ 26
4.2.2細胞激素的基因表現量分析 27
4.2.3免疫後血清中抗體力價 27
4.2.4淋巴球細胞增生試驗 28
第5章 討論 47
參考文獻 . 52
附錄 68
Appendix 1. Example of application of aluminium adjuvant in veterinary vaccine 68
Appendix 2. Antrodia camphorata from solid-state by wood cultivation. 69
Appendix 3. Antrodia cinnamomea (holotype). 70
Appendix 4. The structure of β-1, 3-D-glucan. 71
作者簡介 72

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