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研究生:陳文馨
研究生(外文):Wen-Hsin Chen
論文名稱:α-生育醇在小白鼠及人類皮膚纖維母細胞影響前列腺素新陳代謝之研究
論文名稱(外文):Study of the effects of α-tocopherol on prostaglandin metabolism in mice and human skin fibroblasts
指導教授:麥愛堂麥愛堂引用關係
指導教授(外文):Oi-Tong Mak
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:110
中文關鍵詞:皮膚纖維母細胞α-生育醇前列腺素
外文關鍵詞:human skin fibroblastsα-tocopherolprostaglandin
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前列腺素(prostaglandins;PGs)是調節生理反應進行的重要媒介物質,在其合成過程中經由環氧合酶(cyclooxygenase;COX) 之催化而成;環氧合酶具有兩種同功酶:COX-1及COX-2。COX-1在哺乳動物細胞和組織中為恆定表現,其作用為維持正常生理狀態下前列腺素之合成。而COX-2在正常狀態時為低度表現或不表現,被誘導物刺激時會大量表現。在前列腺素合成反應中,會引發脂質過氧化作用(lipid peroxidation)。過去研究推論脂質過氧化作用被認為是引起心臟血管相關疾病及老化過程的重要原因;補充抗氧化劑,可以抑制脂質過氧化作用之發生。對於補充α-生育醇(α-tocopherol)之抗氧化劑能否抑制由紫外光誘發的前列腺素合成仍不清楚;本研究主要目的在探求補充α-生育醇後對於前列腺素經由自由基氧化代謝之影響,進一步探討此機制與抗老化之相互關係。在本實驗中,以人類皮膚纖維母細胞株及BALB/c小鼠進行體外試驗與動物實驗,分別以不同劑量的紫外光照射處理,再補充不同劑量α-生育醇,接著測定人類皮膚纖維母細胞株與BALB/c小鼠體內脂質過氧化物(malondialdehyde;MDA)含量、催化酶(catalase)、麩胱苷肽過氧化酶(glutathione peroxidase)酵素活性及體內α-生育醇代謝含量變化。為瞭解紫外光照射是否能誘發前列腺素的合成,並造成光老化作用(photoaging)之現象,進一步偵測人類皮膚纖維母細胞株內環氧合酶表現狀況;與小鼠體內前列腺素PGE2、PGF2α、TXA2之代謝情形。實驗結果顯示:補充α-生育醇時,人類皮膚纖維母細胞株及BALB/c小鼠體內α-生育醇代謝含量皆隨之增加,且α-生育醇能降低人類皮膚纖維母細胞株及BALB/c小鼠經由紫外光照射後MDA的含量;對於抗氧化酵素活性方面,α-生育醇能使催化酶與麩胱苷肽過氧化酶活性上升;人類皮膚纖維母細胞株環氧合酶表現以西方墨點分析發現,經由紫外光照射後,能誘導COX-1、COX-2表現量上升;α-生育醇能抑制COX-2的表現。BALB/c小鼠體內前列腺素含量,經由補充α-生育醇後,其含量呈現減少的趨勢。綜合本研究結果,α-生育醇可抑制經由紫外光照射後而誘導的脂質過氧化作用,進而達到抗老化之目的。
Reactive oxygen species(ROS)are shown to be initiating factors in cutaneous aging and many inflammatory disorders. Ultraviolet (UV) radiation induces cell to produce ROS, eicosanoids and malondialdehyde(MDA). Inhibition of these mediators might reduce skin damage. Cyclooxygenase(COX)is believed to be involved in photoaging because of the synthesis of various prostaglandins which are important mediators for inflammation. Antioxidants such as α-tocopherol(Vitamin E)have been found to be photoprotective in animal and in vitro studies. In this study, the effects of α-tocopherol on UV-light induced damage on human skin fibroblasts and BALB/c mice were analyzed by measurements of the cytotoxity, MDA production and enzyme activity of catalase and glutathione peroxidase. Results of both in vitro and in vivo systems showed that treatment with α-tocopherol could prevent depletion of catalase and glutathione peroxidase activity after UV irradiation. Treatment with α-tocopherol also reduced the formation of MDA after UV irradiation. These data demonstrate that human skin fibroblasts respond to UV irradiation stress by antioxidant defense in a similar way as animal system. Furthermore, the effects of α-tocopherol on COX and prostaglandins production in human skin fibroblasts after UV irradiation were also carried out. It was shown that after treatment with α-tocopherol in human skin fibroblasts, the protein levels of COX were reduced after UV irradiation. All these results suggest that α-tocopherol could protect human skin fibroblasts and BALB/c mice against cytotoxic effect of UV irradiation, and the protection might be related to the inhibition of UV induction of lipid peroxidation by COX enzyme.
目錄

目錄…………………………………………………………………………1
圖目錄………………………………………………………………………4
表目錄………………………………………………………………………6
附圖目錄……………………………………………………………………7
縮寫目錄……………………………………………………………………8
中文摘要……………………………………………………………………9
英文摘要……………………………………………………………………11

第一章 前言………………………………………………………………12
(一)、前列腺素的研究歷史及其化學結構…………………………12
(二)、花生四烯酸之代謝路徑………………………………………13
(三)、前列腺素的合成路徑…………………………………………13
(四)、前列腺素生理功能……………………………………………14
(五)、環氧合酶的生化特性…………………………………………16
(六)、抑制劑…………………………………………………………18
(七)、活性氧化物質(ROS)………………………………………19
(八)、抗氧化酵素……………………………………………………19
(九)、脂質過氧化作用………………………………………………20
(十)、紫外光輻射與皮膚老化………………………………………22
(十一)、維生素E功能………………………………………………23
(十二)、維生素E之吸收代謝………………………………………24
(十三)、老化理論……………………………………………………25
(十四)、研究目的……………………………………………………27
(十五)、實驗架構……………………………………………………28

第二章 材料與方法……………………………………………………29
(一)、實驗材料…………………………………………………………29
(二)、實驗儀器…………………………………………………………32
(三)、實驗方法…………………………………………………………34
一 細胞培養…………………………………………………………34
二 細胞繼代培養……………………………………………………35
三 冷凍保存細胞……………………………………………………35
四 細胞存活率………………………………………………………36
五 西方墨點法………………………………………………………36
六 動物飼養…………………………………………………………42
七 血漿中過氧化物含量之測定……………………………………43
八 血液中催化酶 (catalase) 活性測定……………………………43
九 血液中麩胱苷肽過氧化酶 (GSH-Px) 活性測定………………44
十 血漿中α-生育醇之測定…………………………………………44
十一 動物犧牲及樣品收集…………………………………………45
十二 組織均質………………………………………………………46
十三 TXB2的純化……………………………………………………46
十四 PGF2α的純化…………………………………………………47
十五 PGE2的純化……………………………………………………48
十六 前列腺素PGF2α分析…………………………………………48
十七 前列腺素TXB2分析……………………………………………49
十八 前列腺素PGE2分析……………………………………………49
十九 統計分析…………………………………………………………49

第三章 結果………………………………………………………………51
(一)、α-生育醇對於細胞存活率之影響………………………………51
(二)、α-生育醇對於脂質過氧化物之影響…………………………51
(三)、α-生育醇對於α-生育醇代謝之影響.…………………………52
(四)、α-生育醇對於抗氧化酵素活性之影響…………………………52
(五)、α-生育醇對於環氧合酶蛋白質表現之影響……………………52
(六)、α-生育醇對於前列腺素代謝之影響……………………………53
1.PGF2α………………………………………………………………53
2.PGE2………………………………………………………………54
3.TXA2………………………………………………………………54

第四章 討論………………………………………………………………55
(一)、紫外光與α-生育醇對於脂質過氧化物之影響………………55
(二)、紫外光與α-生育醇對於抗氧化酵素活性之影響……………55
(三)、紫外光與α-生育醇對於α-生育醇代謝之影響………………56
(四)、紫外光與α-生育醇對於環氧合酶蛋白質表現之影響………57
(五)、紫外光與α-生育醇對於前列腺素代謝之影響………………58

第五章 結論………………………………………………………………60

第六章 參考文獻…………………………………………………………61
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