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研究生:王思雯
研究生(外文):Ssu-Wen Wang
論文名稱:桑黃中成分 DBL 對於 LPS 誘導 RAW 264.7 細胞之抗發炎路徑探討
論文名稱(外文):Inhibitory effects of DBL from Phellinus linteus on LPS-induced inflammatory response in RAW264.7 macrophages
指導教授:黃冠中黃冠中引用關係
指導教授(外文):Guan-Jhong Huang
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:中國藥學暨中藥資源學系碩士班
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:108
中文關鍵詞:巨噬細胞發炎桑黃
外文關鍵詞:macrophageinflammationPhellinus linteus
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過度或是不受控制的發炎反應已被證實和許多疾病的病程發展相關,包括:類風濕性關節炎、多重硬化症與神經退化性疾病等的一個特殊表徵。故許多實驗致力於尋找出能抑制過度發炎介質物生成,同時能有最低的副作用的成分。
桑黃 (Phellinus linteus) 是一種高經濟效益的保健食品材料,已被證實用具有抗癌症,炎症和過敏的功效。而本實驗將針對桑黃中的成分探討多酚類自然化合物DBL 對細胞膜脂多醣體 (lipopolysaccharide) 誘發老鼠巨噬細胞 RAW 264.7發炎基因表現,產生一連串發炎的影響以及機制的探討。首先藉由 MTT 測定DBL對於細胞的存活率,並發現此藥物並不會對細胞產生毒殺效果。且結果顯示DBL具抑制COX-2 及iNOS 蛋白質表現能力,且當給予DBL後能大幅減少細胞內 TNF-α 及 IL-1β 含量。當更進一步觀察其相關細胞內訊息傳遞分子,其中對象包含 Phosphatidylinositol 3-Kinase (PI3K)-Akt 及三條絲裂原活化蛋白激酶 (MAPK) 路徑。因為許多和發炎有關的基因和細胞結合因子前的啟動子 (promoter) 都具有NF-κB結合區域,NF-κB 活性受其抑制分子 IκB 所調控,因此本實驗也針對IκB/NF-κB 路徑進行探討。結果顯示DBL能有抑制 PI3K/Akt 路徑及 MAPK 家族中p-ERK1/2、p-JNK、p-p38的表現。此外DBL也能有效抑制IKK/IκB的磷酸化及NF-κB進入細胞核。此外,本實驗結果也顯示 DBL 能活化 HO-1 表現,降低細胞的氧化損傷。綜合以上的實驗結果,本論文認為DBL能夠透過抑制LPS而保護細胞達到抗發炎的功效,具有可開發成為抗發炎新生藥物的潛力,期望未來能夠將其真正開發為藥物,為對抗發炎疾病貢獻一份力量。

Excessive or uncontrolled inflammation has been confirmed and progression of many diseases, including: rheumatoid arthritis, multiple sclerosis, and neurodegenerative diseases such as a special characterization. Therefore, many experiments dedicated for searching food ingredients witch can exert inhibition of over-inflammation and have a minimum of side effects of the ingredients.
Phellinus linteus is a highly cost-effective health food material, has been confirmed with the anti-cancer, anti-inflammation and anti-allergic effect. The experiment will explore the polyphenolic natural compounds from Phellinus linteus in DBL. And we use DBL on LPS-induced inflammatory response in RAW 264.7 macrophage cell line. We found DBL didn’t affect the cell viability of RAW 264.7 cell. Moreover, DBL would inhibit the COX-2 and iNOS protein expression, and when given DBL can significantly reduce the intracellular content of TNF-α and IL-1β in a does dependent. Besides, we further observed intracellular signaling molecules, containing phosphatidylinositol 3-Kinase (PI3K)-AKT and mitogen activated protein kinase (MAPK) pathway. And we found DBL inhibited the inflammation via the PI3K/Akt pathway and MAPK family ERK1/2 and c-JNK, p38 protein expression. NF-κB activity is dominated by its inhibiting molecular IκB and upstream signal IKK. Therefore, we also explore IKK/IκB/NF-κB pathway. And our data indicate DBL could suppress IKK and IκB phosphorylation, and increase the NF-κB protein expression in cytoplasm.
Based on the above experimental results, the data indicate that DBL could inhibit LPS induced inflammatory response in vitro. And DBL has capability to develop as an anti-inflammatory newborn drug in the future.

目錄
第一章 研究緣起 1
第二章 文獻回顧 3
第一節 桑黃簡介 3
第二節 藥用成分簡介 7
第三節 發炎反應 8
第四節 RAW 264.7 巨噬細胞 11
第五節 IKK/IκB/NF-κB 12
第六節 Mitogen Activated Protein Kinases(MAPK) 14
第七節 HO-1 16
第三章 實驗方法 17
第一節 實驗架構 17
第二節 研究材料 18
一、 藥品與試劑 18
二、 儀器設備與器材 22
第三節 實驗步驟 24
一、 細胞培養 24
二、 細胞存活率測試 (MTT assay) 27
三、 一氧化碳 ( NO ) 生成分析 29
四、 西方墨點法 ( Western blot ) 31
五、 酵素連結免疫吸附分析法(ELISA) 37
六、 反轉錄-聚合酵素連鎖反應 (RT-PCR ) 41
七、 免疫螢光染色(immunofluorescence staining)46
八、 統計分析 49
第四章 結果 50
第一節 DBL 對於 RAW 264.7 之細胞毒性及存活率之影響 50
第二節 DBL 對 RAW 264.7 細胞產生一氧化氮之影響 52
第三節 DBL 在 RAW 264.7 細胞中影響 LPS 所誘發之 iNOS 及 COX-2 蛋白質表現量 55
第四節 藉由 RT-PCR 探討 DBL 對於 iNOS 及COX-2 的影響 58
第五節 DBL 對 RAW 264.7 細胞之細胞激素影響 60
第六節 DBL 對於 LPS 誘導的 PI3K/Akt 訊息傳遞路徑之影響 62
第七節 在 RAW 264.7細胞中DBL 影響 MAPK 蛋白質表現量 65
第八節 DBL 藉由 IKK/IκB/NF-κB 路徑影響發炎反應 67
第九節 藉由免疫螢光染色法觀察 DBL 對於LPS 誘導的 NF-κB 發炎抑制效果 72
第十節 在 RAW 264.7 中DBL 促進 HO-1 表現 74
第五章 討論 78
參考資料 85

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