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研究生: 蕭嘉羚
論文名稱: 山苦瓜萃取物對人類主動脈內皮細胞表現與分泌細胞黏著分子的影響
指導教授: 吳文惠
學位類別: 碩士
Master
系所名稱: 人類發展與家庭學系
Department of Human Development and Family Studies
論文出版年: 2007
畢業學年度: 95
語文別: 中文
論文頁數: 79
中文關鍵詞: 山苦瓜內皮細胞抗粥狀動脈硬化過氧化體增殖活化受器α
論文種類: 學術論文
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  • 白血球在血管壁內皮細胞上的黏附與滾動是動脈粥狀硬化發生的初期現象,因此,血管內皮細胞分泌之黏著分子、趨化物質等在動脈粥狀硬化形成扮演重要的角色。本實驗室先前發現山苦瓜可降低巨噬細胞PGE2生成,及可減少apoE-/- mice動脈硬化之斑塊面積,本研究為了解苦瓜成分對人類主動脈內皮細胞(human aortic endothelial cells, HAECs)的影響,分別選取兩種品系之山苦瓜,以其乙酸乙酯萃取物(ethyl acetate extract, EAE)、皂化(saponifiable fraction, S)與不皂化(nonsaponifiable fraction, NS)成分與發炎細胞激素-腫瘤壞死因子TNF-α (2 ng/ml) 共同或單獨培養內皮細胞 12 小時,探討山苦瓜對黏著分子 (ICAM-1)、趨化物質 (MCP-1) 、與代表內皮細胞功能的一氧化氮 (NO) 分泌的影響,再與已知苦瓜所含有的純化學成分capric acid、conjugated linolenic acid (CLN)及PPARα ligands藥物fenofibrate相比較,探索苦瓜可能的有效成分。實驗結果發現,山苦瓜乙酸乙酯萃取物均能抑制內皮細胞ICAM-1、MCP-1表現,並減少U937黏附於內皮細胞(p<0.05),但對NO生合成、eNOS之蛋白質與對酵素活化位置Ser1177沒有影響。山苦瓜皂化與不皂化萃取物皆可減少MCP-1表現(p<0.05),但對ICAM-1及NO沒有影響,不皂化萃取物能顯著減少U937的黏附作用(p<0.05)。CLN、capric aicd與fenofibrate可降低MCP-1分泌及單核球黏附作用(p<0.05)。因此推測山苦瓜內含有效成分之一可能為CLN、capric aicd,且除了脂肪酸或脂肪酯化(TG)形式的有效成份之外,應還有其他改善發炎之特殊物質存在於不皂化物萃取物中,這些抗動脈硬化效果可能是透過PPARα調節。整體而言,本研究發現山苦瓜能降低由TNF-α誘導內皮細胞ICAM-1、MCP-1的表現,降低白血球黏結至內皮,此功能與動脈粥狀硬化的降低有關,且此功能並非由苦瓜的單一成分所提供。

    目錄 第一章 緒論...............................1 第二章 文獻探討...........................2 第一節 動脈粥狀硬化的病理過程............2 一、內皮細胞受損反應假說..................2 二、黏著分子 ............................4 三、MCP-1(Monocyte chemotactic protein-1單核球趨化蛋白-1) ..........................................5 四、TNF-α(tumor nerosis factorα 腫瘤壞死因子α)...6 五、 NO(nitric oxide,一氧化氮)與eNOS(endothelial nitric oxide synthase,內皮行一氧化氮合成酶).....6 六、NF-κB(Nuclesr factor-κB,核因子-κB)...9 第二節 PPAR.............................11 一、過氧化體增殖劑活化受器PPAR...........11 二、PPARs與動脈硬化的相關性..............12 第三節 苦瓜介紹..........................14 一、名稱與植物性狀.......................14 二、生產分布與栽培環境...................15 三、食用特性 ...........................15 四、山苦瓜介紹 ..........................15 五、苦瓜生理效用.........................17 第四節 研究動機與目的....................18 第三章 材料與方法........................19 第一節 細胞培養.........................19 第二節 山苦瓜樣品製備...................20 一、乙酸乙酯萃取物製備(ethyl acetate extract;EAE)...20 二、皂化與不皂化萃取物(Saponifiable fraction or substances ;S and Non-saponifiable fraction or substances;NS)...20 三、已知苦瓜之純化學成分及PPARα ligand正對照藥物...21 第三節 實驗設計...............................22 第四節 MTT細胞存活率分析......................23 第五節 MCP-1分析..............................23 第六節 ㄧ氧化氮分析...........................24 第七節 黏著分子ICAM-1分析.....................24 第八節 eNOS蛋白質與活化分析...................27 第九節 內皮細胞/單核球黏附分析................29 第十節 山苦瓜中CLN含量分析....................29 第十一節 統計分析.............................29 第四章 結果....................................30 第一節 HAEC細胞培養條件之建立.................30 第二節 樣品對細胞存活之影響 ..................31 第三節 山苦瓜乙酸乙酯萃取物(EAE)對內皮細胞表現ICAM-1、MCP-1的影響........................................38 一、黏著分子ICAM-1蛋白質表現...................38 二、單核球趨化蛋白MCP-1濃度....................38 第四節 山苦瓜皂化萃取物(S)對內皮細胞表現ICAM-1、MCP-1的影響.............................................43 一、黏著分子ICAM-1蛋白質表現...................43 二、單核球趨化蛋白MCP-1濃度....................43 第五節 山苦瓜不皂化萃取物(NS)對內皮細胞表現ICAM-1、MCP-1的影響...........................................48 一、黏著分子ICAM-1蛋白質表現...................48 二、單核球趨化蛋白MCP-1濃度....................48 第六節 CLN、capric acid及fenofibrate對內皮細胞表現ICAM-1、MCP-1的影響....................................53 一、黏著分子ICAM-1蛋白質表現...................53 二、單核球趨化蛋白MCP-1濃度....................53 第七節 山苦瓜對TNF-α誘導之內皮細胞黏附U937的影響.......56 第八節 山苦瓜對內皮細胞NO生合成及eNOS蛋白質表現的影響...58 一、一氧化氮NO.................................58 二、內皮細胞一氧化氮合成酶eNOS.................58 (一)eNOS蛋白質表現...........................58 (二)eNOS酵素活化.............................58 第九節 山苦瓜中CLN含量估計....................63 第五章 討論....................................64 第一節 山苦瓜萃取物對ICAM-1、MCP-1的影響......64 第二節 山苦瓜乙酸乙酯萃取物對一氧化氮生合成與eNOS的影響...65 第三節 山苦瓜對U937黏附於內皮細胞的影響.......65 第四節 山苦瓜中之有效成分評估.................66 第五節 山苦瓜可能作用機制之探討...............67 第六節 結論 ..................................69 參考文獻.......................................71

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