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研究生:黃芷渝
論文名稱:茶黃素影響巨噬細胞活化機制之探討
論文名稱(外文):Study on the Mechanism of Theaflavin-3,3'-digallate Affects Macrophage Activation
指導教授:林淑美林淑美引用關係
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:食品科學系研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
畢業學年度:102
語文別:中文
中文關鍵詞:痛風巨噬細胞茶黃素
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痛風為常見代謝性關節炎,與嘌呤代謝異常導致高尿酸血症及尿酸鹽沉積於關節腔有密切關聯。尿酸鹽結晶沉積引發發炎反應並導致關節組織結構損傷。急性發炎期常伴隨大量嗜中性細胞浸潤至關節。尿酸鹽結晶誘發發炎反應之分子機制尚未十分明確。巨噬細胞是參與先天性免疫反應的重要細胞,依誘發環境不同而有功能特異的M1或M2型細胞。病原微生物感染或IFN-刺激下促進M1型細胞的極化並主導發炎反應,而M2型細胞則與組織清除、修復,以及組織恆定有關。然而巨噬細胞在尿酸鹽沉積誘導痛風性發炎機制的角色則仍待釐清。目前急性痛風性關節炎的臨床治療多使用秋水仙素及非固醇類抗發炎藥物舒緩發炎症狀,並以allopurinol降低血液中尿酸濃度預防復發。而藥物治療多伴隨副作用產生,且痛風發生率常與飲食有關,因此從天然食材中尋找具有抗發炎及抗痛風的植物營養素 (phytonutrients) 以輔助或取代藥物使用渴望成為未來趨勢。茶黃素為紅茶的主要功能性成份,已知具有抗氧化、抗發炎、抗肥胖、抗癌及調整血糖等多種保健功能。本實驗進一步探討茶黃素影響巨噬細胞活化之機制。以尿酸鹽結晶誘發RAW 264.7小鼠巨噬細胞為實驗模式,並與脂多醣誘發之典型發炎反應做比較。結果顯示巨噬細胞具清除細胞外尿酸鹽結晶的作用,並於細胞質內形成空泡,而此細胞型態改變隨著尿酸鹽結晶處理時間的延長而趨向回復至正常細胞型態,此與脂多醣造成細胞質突出形成僞足的型態不同。而尿酸鹽結晶與脂多醣對巨噬細胞典型發炎反應指標之影響亦不盡相同,尿酸鹽結晶誘發的巨噬細胞並不釋放發炎物質:一氧化氮及介白素1 beta,但影響腫瘤壞死因子alpha及單核細胞趨化蛋白1的表現及分泌,同時提高過氧化物體增殖物啟動受體γ (peroxisome proliferation activating receptor-gamma; PPAR-gamma) 及細胞表面清除受體CD36的基因表現。再者,尿酸鹽結晶並不會誘發M1巨噬細胞極化作用,反而提高M2細胞指標基因CD206及精氨酸酶1的表現。而且本研究中的尿酸鹽結晶處理Raw264.7細胞模式不會誘發基質金屬蛋白酶基因表現及酵素活性。而茶黃素的介入可降低尿酸鹽結晶所誘發腫瘤壞死因子、單核細胞趨化蛋白1及PPAR-gamma基因表現,並提高CD36接受器基因表現,此與加速巨噬細胞清除尿酸鹽結晶作用有關,藉此機制TF3可能達預防痛風性關節炎之功效。另外,本研究也證實TF3之抗發炎機制與抑制M1巨噬細胞極化途徑有關,值得進一步探討。
中文摘要 I
ABSTRACT III
目錄 V
圖目錄 VII
壹、前言 1
貳、文獻回顧 3
一、痛風病理機制 3
二、痛風性發炎反應 5
三、基質金屬蛋白酶與痛風 8
四、痛風性關節炎治療用藥 9
五、痛風之替代療法 10
參、材料與方法 12
實驗細胞株與樣品 12
實驗器材及耗材 12
實驗藥品 13
PCR引子 (primer) 序列及產物分子量 16
基因偵測之PCR條件 18
細胞株培養 19
冷凍細胞活化 19
細胞冷凍保存 19
細胞繼代 20
細胞實驗處理 20
細胞存活率試驗 20
一氧化氮 (nitric oxide, NO) 含量測定 21
細胞型態觀察 22
巨噬細胞極化之細胞表面抗原偵測 22
反轉錄聚合酶鏈鎖反應 (reverse transcription polymerase chain reaction, RT-PCR) 23
細胞激素含量測定 25
明膠蛋白酶活性電泳分析 (gelatin zymography) 25
即時聚合酶鏈鎖反應 (real-time polymerase chain reaction) 26
統計分析 27
肆、實驗架構 28
伍、結果 29
一、單鈉尿酸鹽結晶之巨噬細胞毒性檢測 29
二、茶黃素之RAW 264.7巨噬細胞毒性檢測 29
三、茶黃素對尿酸鹽結晶誘發RAW 264.7巨噬細胞發炎反應之影響 30
四、 茶黃素對尿酸鹽結晶誘發RAW264.7巨噬細胞表型轉換 (M1/M2 polarization) 之影響 34
五、茶黃素對尿酸鹽結晶誘發RAW 264.7巨噬細胞型態之變化 35
六、 茶黃素對尿酸鹽結晶誘發RAW 264.7巨噬細胞基質金屬蛋白酶9 (MMP-9) 之影響 37
陸、討論 65
柒、結論 73
捌、參考文獻 76
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