樹突細胞為抗原呈現細胞 (Antigen Presenting Cells, APC) 作為先天免疫及後天免疫之橋梁，因此樹狀細胞被認為是在免疫調控發展過程中的一個主要調控對象。而本計畫主要探討巴豆苷 (crotonoside) 於受脂多醣體 (Lipopolysaccharide, LPS) 活化小鼠骨髓轉化樹狀細胞之作用及其治療於類風溼性關節炎小鼠之功效。而結果發現crotonoside能抑制LPS誘導樹突細胞所分泌的細胞激素，TNF-α、IL-6、IL-12 p40以及一氧化氮釋放和活性氧的表現，同時也降低了表面共刺激分子CD80和CD86。進一步探討其分子機制，研究發現crotonoside能抑制LPS誘導的MAPKs-p38及ERK磷酸化並減少NF-κB入核的情形。而探討crotonoside於第二型膠原蛋白誘導關節炎小鼠治療之功效實驗，結果發現crotonoside可減緩關節炎小鼠關節腫脹的情形，並且減少淋巴結細胞中功效性Th1細胞及增加調節性Treg細胞的比例。總結來說，此次的結果首度發現crotonoside能調節樹突細胞活化並且在關節炎小鼠模式上具有減緩其病症之功效，結果顯示crotonoside具有發展成為治療人類類風濕性關節炎的藥物潛力。

Dendritic cells are antigen-presenting cells (APCs) as a bridge between innate immunity and adaptive immunity, therefore dendritic cells are considered to be a major regulatory target in the development of immunomodulatory. This study is mainly to investigate the effect of crotonoside on the lipopolysaccharide (LPS) induced maturation of murine bone marrow-derived dendritic cells (BMDCs) and treatment collagen induced arthritis mice. This result discovered that crotonoside inhibits LPS-induce cytokines secreted by BMDCs, TNF-α, IL-6, IL-12 p40, and the release of nitric oxide and reactive oxygen species. At the same time, crotonosdie were also reduction the co-stimulatory molecules CD80 and CD86. Further confirming its molecular mechanism, crotonoside can inhibit the activation of MAPKs-p38 and ERK induced by LPS-stimulated BMDCs and inhibit NF-κB pathway into the nucleus. Further investigate its molecular mechanism, crotonoside can inhibit the activation of MAPKs induced by LPS-stimulated BMDCs and inhibit NF-κB pathway into the nucleus. To investigate the effect of crotonoside in the treatment of type 2 collagen-induced arthritis mice, it was found that crotonoside can slow the joint swelling of arthritic mice and reduce the proportion of Th1 cells and increase level of regulatory Treg cells in lymph node cells. In summary, this result was first time found that crotonoside can regulate dendritic cell maturation and has the efficacy in the arthritic mouse model, suggesting that crotonoside has the potential to develop into a drug for the treatment of human rheumatoid arthritis.

第一章、緒論 1
1. 免疫系統中的樹突細胞 (Dendritic cell in immune system) 1
2. 自體免疫疾病 (Autoimmune disease, AID) 3
3. 類風溼性關節炎 (Rheumatoid arthritis, RA) 4
4. 樹突細胞與類風溼性關節炎之橋樑 4
5. 巴豆植物-天然化合物糖苷類 5
6. 研究動機 (Motivation) 6
7. 實驗架構 6
第二章、材料方法 7
1. 小鼠骨髓分化樹突細胞培養 7
2. 細胞存活率分析 7
3. 細胞激素釋放分析 (Enzyme-linked immunosorbent assay, ELISA) 8
4. 一氧化氮檢測 8
5. 細胞表面抗原之分析 9
6. 活性氧能力分析 9
7. 西方墨點法 10
8. 核質分離及NF-κB p65 活性分析 12
9. 第二型膠原蛋白誘導之關節炎小鼠模式 (Collagen-induced arthritis) 12
10. 血清抗體分析 14
11. 細胞內細胞激素染色分析 14
12. 統計分析 15
第三章、 實驗結果 16
1. 評估天然化合物於受到LPS刺激之小鼠骨髓分化之樹突細胞的免疫調節能力 16
2. 評估天然化合物對於樹突細胞的細胞毒性 17
3. 評估Crotonoside對於樹突細胞的免疫調節可能性 17
4. Crotonoside能抑制LPS誘導樹突細胞活化所表現的共激分子 17
5. Crotonoside能抑制LPS誘導樹突細胞活化所表現活性氧的能力 18
6. Crotonoside能抑制LPS誘導樹突細胞活化所表現MAPK, NF-κB 18
7. Crotonoside能減緩第二型膠原蛋白誘導關節炎的小鼠模式 18
8. Crotonoside不會影響第二型膠原蛋白特異性IgG的表現量 19
9. Crotonoside能增加關節炎小鼠淋巴結內調節性T細胞的數目 (Treg) 並且減少CD4+ T細胞IFN-ϒ之表現 19
第四章、 討論 20
第五章、 參考文獻 23
第六章、圖表 28
圖一、分析天然物在LPS誘導之小鼠骨髓分化樹突細胞的免疫調節能力 28
圖二、Crotonoside於小鼠骨髓分化之樹突細胞的毒性影響 29
圖三、 Crotonoside 抑制LPS刺激小鼠骨髓誘導樹突細胞的免疫調節能力 30
圖四、抑制LPS刺激小鼠骨髓誘導樹突細胞活化所表現之共激分子 31
圖五、Crotonoside能抑制LPS刺激小鼠骨髓誘導樹突細胞活化所表現活性氧的能力 32
圖六、Crotonoside能抑制LPS刺激小鼠骨髓誘導樹突細胞活化時MAPK、p-JNK及NF-κB活化路徑 34
圖七、Crotonoside能減緩第二型膠原蛋白誘導關節炎的小鼠模式 36
圖八、Crotonoside能增加關節炎小鼠淋巴結CD4+T細胞CD25且減少CD4+ T細胞IFN-ϒ之表現 38

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