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研究生:吳珮儀
研究生(外文):Pei-Yi Wu
論文名稱:一氧化碳誘導環氧化酶乙表現之分子機制探討
論文名稱(外文):Study of the molecular mechanisms of carbon monoxide on the induction of cyclooxygenase-2 in macrophage and microglia
指導教授:梁有志梁有志引用關係
指導教授(外文):Yu-Chih Liang
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學技術學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:86
中文關鍵詞:環氧化酶-2血紅素氧化酶巨噬細胞一氧化碳
外文關鍵詞:cyclooxygenase-2heme oxygenasemacrophagecarbon monoxide
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血紅素氧化酶 (heme oxygenase) 是將血紅素 (heme) 降解產生膽綠素/膽紅素、亞鐵離子和一氧化碳 (carbon monoxide)。血紅素氧化酶的許多生理功能都與其副產物一氧化碳有關,在許多細胞中,如其具有抗氧化、血管舒張、影響週邊或中樞神經系統的神經傳導活性、抗發炎、抗細胞凋亡或者抗細胞增殖等功能。一氧化碳釋出分子 (carbon monoxide-releasing molecules) 是一類新興藥物試劑,在生物系統中,釋出的一氧化碳可以去調節細胞功能。本篇的研究中,在微小膠質細胞 (microglia) 和巨噬細胞 (macrophage) 中,利用一氧化碳釋出分子 (CO-RMs) 和一氧化碳氣體 (CO gas) (500ppm) 去檢測環氧化酶-2 (cyclooxygenase-2, COX-2) 的表現。從西方墨點法 (Western blot) 和反轉錄酶-聚合酶連鎖反應 (reverse transcriptase-polymerase chain reaction, RT-PCR) 實驗證實,在有脂多醣(Lipopolysaccharide, LPS)刺激的微小膠質細胞(BV2 and EOC13.31)和巨噬細胞 (RAW264.7) 中,CO-RMs和CO gas可以抑制一氧化氮合成酶(inducible nitric oxide synthase, iNOS) 蛋白質和mRNA的表現。然而,細胞無論是否受到LPS的刺激,CO-RMs和CO gas都會增加COX-2的表現。在訊息傳遞方面,CO-RMs會誘導MAPKs及Akt磷酸化,且隨著時間的增加磷酸化的表現也就愈明顯。另外,加入了PKG、p38、Erk、JNK抑制劑後,會抑制掉COX-2的表現。根據實驗結果得知,CO誘導COX-2的表現可能是經由PKG、p38、Erk和JNK的路徑來傳達。我們更進一步想要探討,在初級腦皮質細胞中經由CO處理而誘導COX-2表現是否是促成神經細胞死亡的原因。
The enzyme heme oxygenase (HO) degrades heme to produce biliverdin/bilirubin, ferrous iron and carbon monoxide (CO). Many biological functions of HO have been attributed to its enzymatic byproduct carbon monoxide (CO) that exhibits anti-oxidative, vasodilation, and neurotransmission activities in the central or peripheral nervous system, as well as anti-inflammatory, anti-apoptotic, or anti-proliferative potential in many cells. Carbon monoxide-releasing molecules (CO-RMs) are emerging as a new class of pharmacological agents that regulates important cellular function by liberating CO in biological system. In this study, we used both CO-RMs and CO gas to examine the regulation of cyclooxygenase-2 (COX-2) expression in microglia/macrophage. Western blot and RT-PCR analysis demonstrated that CO-RMs and CO gas (500 ppm) significantly inhibited the protein and mRNA expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-activated (BV2 and EOC13.31) microglia and (RAW264.7) macrophage. However, CO-RMs and CO gas up-regulated COX-2 expression in the cells with or without LPS. CO-RMs time-dependently induced the phosphorylation of MAPKs and Akt. In addition, the induction of COX-2 could be reversed by PKG, p38, Erk and JNK inhibitors. The results suggest that the induction of COX-2 expression by CO might mediate PKG, p38, Erk and JNK. Further works are to investigate whether induction of COX-2 contribute to the neuron death in primary cortical cells exposed to CO.
中文摘要 (Abstract in Chinese) I
英文摘要 (Abstract in English) II
目錄 III
圖表目次 (Figures lists) VI
縮寫表(Abbreviations) VIII
緒論(introduction) 1
壹、一氧化碳 (Carbon monoxde) 1
1、一氧化碳簡介 1
2、內生性一氧化碳的來源 2
3、一氧化碳與腦神經組織 3
(1) 腦神經組織介紹 3
(2) 腦神經組織發炎反應 4
4、一氧化碳與抗發炎反應 5
5、一氧化碳與抗細胞凋亡 ( anti-apoptosis) 6
6、一氧化碳與抑制細胞增殖 (anti-proliferative) 7
貳、一氧化碳的訊號傳遞相關路徑 7
參、環氧化酶-2 (Cyclooxygenase-2; COX-2) 8
1、簡介 8
2. COX-2功能與應用 11
3.訊號傳遞相關路徑 13
肆、一氧化碳釋出分子-2 (Carbon monoxide-releasing molecules, CORM-2) 15
1、CORM-2 簡介 15
2、CORM-2 功能與應用 16
實驗材料與方法 17
壹、實驗材料 17
1、混合氣體(CO 500 ppm、5% CO2、Air) 17
2、Tricarbonyldichloro ruthenium (II) dimmer [Ru(CO)3Cl2]2 ; CORM-2 17
3、Ruthenium (III) chloride hydrate [Ru(DMSO)3Cl2]2 ; RuCl 17
4、藥品試劑 17
5、儀器 18
貳、細胞培養實驗 : 19
1、EOC13.31: 19
2、EOC細胞培養液的製備: 20
參、微小膠質細胞的培養 : 20
肆、RAW264.7 巨噬細胞的培養 21
伍、西方墨點法 (Western blot) 21
1、細胞蛋白質之收集 21
2、蛋白質含量測定 22
3、1級抗體及2級抗體 22
4、實驗方法 23
陸、反轉錄酶-聚合酶連鎖反應 (Reverse Transcriptase- polymerase Chain Reaction, RT-PCR) 24
1、萃取核糖核酸 (RNA) 24
2、反轉錄 (Reverse Transcription) 24
3、聚合酶連鎖反應 (Polymerase chain reaction) 24
柒、一氧化碳氣體實驗 : 25
附錄 26
實驗結果 (Results) 28
壹、一氧化碳氣體 (500 ppm) 抑制了微小膠質細胞 (BV-2)及巨噬細胞 (RAW264.7)中LPS所誘導iNOS蛋白質和mRNA的表現,但加強了COX-2蛋白質及mRNA的表現。 28
(1) 微小膠質細胞 28
(2) 巨噬細胞 28
貳、Carbon monoxide-releasing molecules (CORM-2) 誘導微小膠質細胞(EOC13.31)與巨噬細胞(RAW264.7)表現COX-2蛋白質的表現 29
(1) 微小膠質細胞及巨噬細胞 29
(2) CORM-2與CO gas (500 ppm)誘導初級微小膠質細胞中COX-2蛋白質表現 29
參、CORM-2抑制了微小膠質細胞(BV-2)及巨噬細胞 (RAW264.7)中LPS所誘導iNOS蛋白質和mRNA的表現,卻增強了COX-2蛋白質及mRNA的表現。 30
(1) 微小膠質細胞 30
(2) 巨噬細胞 30
肆、CORM-2在巨噬細胞中的訊號傳遞路徑 30
(1) CORM-2 所誘導的p-Akt活性表現的關係 31
(2) CORM-2 所誘導的p-JNK活性表現的關係 31
(3) CORM-2 所誘導的p-Erk活性表現的關係 31
(4) CORM-2 所誘導的p-p38活性表現的關係 31
(5) CORM-2 所誘導的p-IkBa活性表現的關係 32
伍、MAPK及Akt抑制劑可抑制巨噬細胞中CORM-2所誘導COX-2蛋白質表現 32
(1) JNK抑制劑 32
(2) Erk抑制劑 32
(3) p38抑制劑 33
(4) Akt抑制劑 33
陸、PKG抑制劑 (KT5823) 抑制巨噬細胞中CORM-2所誘導COX-2蛋白質表現 34
討論 (Discussion) 35
壹、CORM-2與CO Gas抑制iNOS表現,但增強COX-2表現 35
貳、CORM-2誘導COX-2表現的訊號傳遞路徑 36
參、COX-2表現對於微小膠質細胞的影響 38
肆、結論 40
參考文獻 (References) 41
結果圖表 55
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