臺灣博碩士論文加值系統

中文摘要
微膠細胞活化已被廣泛證實介導的炎性過程是至關重要的幾個神經退行性疾病。藥物可以在微膠細胞提供直接的抑制作用，因此被認為是抗衡神經退行性進展的潛在戰略。咖啡酸苯乙酯(caffeic acid Phenethyl Ester； CAPE)，在蜜蜂蜂膠天然的酚類，是已知的具有抗氧化和抗發炎的特性。因此，目前的研究旨在通過使用體外和體內模型來探測CAPE對微膠細胞活化的影響。結果：western bolt或Griess試劑測定顯示CAPE顯著抑制誘導型一氧化氮合酶(inducible nitric oxide synthase； iNOS)，環氧合酶-2(cyclooxygenase-2； COX-2)和一氧化氮(nitric oxide； NO)的表達。咖啡酸的刺激導致血色素氧化酵素（Hemeoxygenase； HO-1），促紅血球生成素(Erythopoietin； EPO)和細胞因子信號傳導(Suppressor of Cytokine Signaling； SOCS3)在微膠細胞中的蛋白增加。磷酸腺苷活化蛋白激酶-α(activated protein kinase α； AMPK-α)進一步發現，調節CAPE的抗炎作用。從免疫組織化學實驗的結果也顯示CAPE的微膠細胞活化的抗神經炎效果。結論：目前的研究已證明幾種可能的決定分子，EPO 、AMPK、HO-1和SOCS3，在微膠細胞抗神經炎反應當中作為細胞保護因子。
Keywords：iNOS、COX-2、NO、EPO、HO-1、AMPK、SOCS3、CAPE

英文摘要
Microglial activation has been widely demonstrated to mediate inflammatory processes that are crucial in several neurodegenerative disorders. Pharmaceuticals that can deliver direct inhibitory effect on microglia is therefore considered as a potential strategy to counterbalance neurodegenerative progression. Caffeic acid phenethyl ester, a natural phenols in honeybee propolis, is known to possess antioxidant, and anti-inflammatory properties. Accordingly, the current study intended to probe the effects of CAPE on microglia activation by using in vitro and in vivo models. Results: western blots or Griess reagent assay revealed CAPE significantly inhibited the expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and the production of nitric oxide (NO). Administration of caffeic acid resulted in increased expressions of hemeoxygenase (HO-1), erythropoietin (EPO) and suppressors of cytokine signaling (SOCS3) in microglia.The phosphorylated adenosine monophosphate-activated protein kinase-α (AMPK-α) was further found to regulate the anti-inflammatory effects of CAPE. In vivo results from immunohistochemistry along with rotarod test also revealed the anti-neuroinflammatory effects of CAPE in microglia activation. Conclusion: The current study has evidenced several possible molecular determinants, EPO, AMPK, HO-1 and SOCS3, in mediating anti-neuroinflammatory responses in microglial cells.

縮寫表...........................................................................................................4
中文摘要………………………………………………………………..….5
英文摘要……………………………………………………………..…….6
第一章 前言……………………………………………………………….7
第一節 研究背景……………………………………………………...7
1.1.1 微膠細胞 (microglia cell)…………………………………7
1.1.2 促發炎與抗發炎反應 (pro-inflammation and anti-inflamm
ation)……………………….……………………….……...8
1.1.3 咖啡酸苯乙酯 (caffeic acid phenethyl ester)………..……9
1.1.4 促紅血球生成素 (erythropoietin；EPO)………………..11
1.1.5 血紅素氧化酵素 (hemeoxygenase；HO)………………11
1.1.6 細胞因子信號抑制劑 (suppressor of cytokine signaling, SOCS)……………………………………...……………....12
第二節 研究目的…..…………………………………………...…...20
第二章 研究方法……………………………………………………..…21
第一節 研究材料……………………………………………………21
2.1.1 實驗細胞株 (cell line)…………………………………...21
2.1.2 細胞培養 (cell culture)…………………………………..21
2.1.3 西方墨點法 (western boltting)…………………………..21
2.1.4反轉錄聚合酶連鎖反應(reverse transcriptasepolymerase chain reaction)………………………….………………......22
2.1.5 抗體 (antibody)…………………………………………..22
2.1.6 抑制劑 (inhobitor)……………………………………….23
第二節 實驗方法……………………………………………………24
2.2.1 細胞培養 (cell culture)……………….…...………..........24
2.2.2 細胞相對存活率檢測 (cell viability assay)……………..24
2.2.3 一氧化氮測定法 (assay of nitric oxide)…………….…..24
2.2.4 RNA之萃取 (RNA extraction)……………………….…25
2.2.5 反轉錄聚合酶連鎖反應 ( reverse transcriptasepolymerase chain reaction)………………………...…………………...25
2.2.6 Quantitative real time-PCR……………………………....26
2.2.7 西方墨點法 (western blotting)……………………….…27
2.2.8 免疫組織化學染色..…………………………………......27
2.2.9 運動協調試驗 (Motor coordination)…………….…...…28
2.2.9 統計方法…………………………………………….…...28
第三章 研究結果……………………………………………………………………………………….………29
3.1 CAPE在微膠細胞中抑制iNOS和COX-2和其他發炎因子 合成機轉之探討…..……………………….…………...…29

3.2 CAPE對LPS刺激微膠細胞發炎反應的調節途徑與機轉29
3.3 CAPE增加細胞保護因子HO-1、SOCS-3、EPO在抗發炎反應上的表現……………………………...........................30
3.4 CAPE誘導AMPK、PKC的增加而探討其細胞保護因子
HO-1、SOCS-3、EPO的相關途徑…………………….....30
3.5 在動物實驗中，CAPE抑制LPS刺激微膠細胞活化，
改善小鼠運動功能障礙…………………………………....31
第四章結論與討論………………………………………………………33
參考文獻………………………………………………...……………….46

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