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研究生:李傳旺
研究生(外文):Lee Chwan Wang
論文名稱:維生素D3對內毒素刺激膠質細胞產生一氧化氮、細胞素及活性氧化物質之抑制作用:與其他抗氧化藥物之比較
論文名稱(外文):Inhibition Effects of Vitamin D3 on the Production of Nitric Oxide, Cytokine and Reactive Oxygen Species in Lipopolysacharide-stimulated Glial Cells: a Comparison with other Anti-Oxidants
指導教授:王家儀王家儀引用關係
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
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
中文關鍵詞:內毒素細胞素膠質細胞活性氧化物質
外文關鍵詞:LipopolysaccharideLPSCytokineGlial cellsReactive oxygen species
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1,25-dihydrovitamin D3 (Vit.D3) 為維生素D的主要代謝活性衍生物,其生理作用可調節體液鈣離子及磷酸鹽的恆定,已有研究證實維生素D的接受器存在於大腦中,並可降低神經細胞的氧化性傷害及誘導膠質細胞產生神經滋養因子。許多神經退化性疾病,都會產生發炎反應,而膠質細胞 (包括星狀細胞及微小膠質細胞) 被認為在免疫和發炎反應中扮演重要角色,當神經受損時,會活化膠質細胞,並釋出發炎物質,包括一氧化氮 (nitric oxide, NO),活性氧物質 (reac tive oxygen species, ROS) 及腫瘤壞死因子 (tumor necrosis factor α, TNFα)。許多文獻均顯示內毒素 (lipopolysaccharide, LPS) 可活化膠質,藉由活化 (nuclear factor —kappa B, NFκB) 而誘導一氧化氮合成酶 (inducible nitric oxide synthase, iNOS) 與細胞素之基因與蛋白質表現。近來研究指出活性氧物質在NFκB的活化過程中扮演細胞內訊號傳遞的角色,在本研究中,我們以LPS處理初代培養的混合膠質細胞,作為活化膠質細胞之實驗模式,並合併給予Vit.D3或ROS清除劑,包括超氧自由基歧化酶 (superoxide dimutase, SOD)、雙甲基連素硫 (dimethylthiourea, DMTU) 與調節細胞內氧化還原狀態之藥物,包括還原態麩胺基硫 (reduced glutathione, GSH)、N-acetylcysteine (NAC)、aminooxyacetic acid (AOAA) 及buthionine sulfoximine (BSO),與抗氧化劑 (vitamin C), 以探討細胞內氧化還原狀態是否影響ROS的生成、與NO、TNFα釋放並比較各藥物之抑制效果。
我們以2,7-chlorofluorescin diacetate (DCF-DA) 測量法,測量細胞內ROS的產生量、以Griess反應法測量培養液中nitrite累積量來評估NO釋放量,以酵素連結免疫分析法 (enzyme linked immunoadsorbent assay) 測量培養液中TNFα之濃度,實驗結果顯示LPS可以刺激膠質細胞產生NO、ROS及TNFα,且此作用與LPS之濃度及處理之時間有關 (concentration and time dependent),Vit.D3、AOAA及Vit.C有濃度依賴性抑制LPS所誘導的NO釋放。LPS誘導ROS的表現量在18小時達最高量,Vit.D3、SOD、AOAA及Vit.C可減少ROS的產生。LPS誘導TNFα的釋放,在6小時近乎最高量,Vit.D3、AOAA、 NAC、BSO能明顯抑制TNFα的釋放。以上結果顯示Vit.D3在低於μM的濃度 (5x10-8M) 即能明顯抑制膠質細胞釋放發炎物質,Vit.D3較其他抗氧化藥物更強烈抑制LPS所誘導膠質細胞的ROS生成與NO、TNFα的釋放,或許Vit.D3能抑制神經損傷或遭受感染時膠質細胞的活化。

1,25-dihydroxyvitamin D3 (or Vit.D3), an active metabolite of vitamin D, is well known for its systemic regulatory role in the maintenance of calcium homeostasis. The receptors for Vit.D3 (VDR) are found in the CNS. Recent evidence also indicates its neuroprotective and antioxidant potential in the central nervous system (CNS).
Glial cells are centrally involved in inflammatory and immunological responses in pathological conditions including neurodegenerative disease, ischemia and trauma. Neuronal injury is an activation of glial cells (reactive astrocytes or microglia), resulting in the production of reactive oxygen species (ROS), nitric oxide (NO) and the release of proinflammatory cytokines such as tumor necrosis factor-* (TNF-*).
Because ROS mediate the signal for NFκB activation in glial cells, we examined the role of vitamin D3 acted as an antioxidant and compared with ROS scavengers, including superoxide dismutase (SOD), dimethyl and thiourea (DMTU), redox regulators, including reduced glutathione (reduced GSH), N-acetylcysteine (NAC), aminooxyacetic acid (AOAA) and buthionine sulfoximine (BSO), and antioxidants such as vitamin C, in the production of ROS induced by LPS.
In this thesis, glial activation by LPS was used as models to investigate the role of ROS and redox status in the expression of iNOS and cytokines induced by LPS using primary culture of mixed glial cells. The expression of iNOS was estimated by measuring NO production using the Griess method for nitrite, a stable metabolite of NO. Quantitation of cytokines (TNF-*) was measured by enzyme linked immunosorbent assays (ELISA). ROS production was measured by 2,7-chlorofluorescin diacetate (DCF-DA).
LPS elevated nitrite levels in a concentration-dependent manner, Vit.D3, AOAA and Vit.C concentration-dependently inhibited the elevation of nitrite levels by LPS.
LPS increased intracellular ROS production which reached the maximum at 18 hours. Vit.D3, SOD and AOAA suppressed the ROS production. LPS increased TNFα near maximal levels following treatment for 6 hours. Vit.D3, AOAA, NAC and BSO decreased LPS-induced TNFα release from mixed glia.
These results demostrate that Vit. D3 suppresses the production of NO, TNFα and ROS in LPS-stimulated glial cells, and more effective than other drug used in this study, suggesting that Vit. D3 may suppress the activation of glial cells during inflammatory reactions elicited by CNS infections or injuries.

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目錄¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨ Ⅰ
圖目錄¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨ Ⅱ
表目錄¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨ Ⅴ
中文摘要¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨ Ⅵ
英文摘要¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨ Ⅷ
第一章 緒論¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨ 1
第二章 材料與方法¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨16
第三章 實驗結果¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨ ¨¨ 24
第四章 討論¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨ 34
第五章 結論¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨ 43
第六章 參考文獻¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨¨ 44

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