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研究生:湯銘吉
論文名稱:CyclicAMP調控肺泡巨噬細胞釋放一氧化氮和TNF-a的機轉探討
論文名稱(外文):Regulation of nitric oxide and TNF-a production by cyclic AMP in alveolar marcophages
指導教授:黃聰龍黃聰龍引用關係
學位類別:碩士
校院名稱:長庚大學
系所名稱:天然藥物研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
中文關鍵詞:肺胞巨噬細胞NR8383cyclic AMPNOTNF-alphaiNOSLPS
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肺胞巨噬細胞 (AM)主要是擔任肺部防禦的工作,能有效抵抗空氣中外來物質的侵害,如粉塵、微生物及環境毒素。實驗上,使用NR8383與primary alveolar macrophage (PAM)二種巨噬細胞來研究cAMP和磷酸二酯酶 (PDE)在NO與TNF-α釋放上的作用。結果顯示,cAMP的類似物,dbt-cAMP與8br-cAMP,以時間和濃度相關性的增加NO釋放與iNOS表現。PDE的活性可被PDE3與PDE4抑制劑所抑制,然而PDE4抑制劑比PDE3抑制劑更能加強PGE1和forskolin誘導的NO釋放與iNOS表現; 在cAMP含量與CREB磷酸化上也可得到相同的加強作用。另外,PKA抑制劑能減少cAMP誘導的NO釋放和CREB磷酸化。由這些結果可以推測,當肺泡巨噬細胞在受刺激而合成cAMP時,抑制PDE4可能比抑制PDE3更能有效的增加NO形成。在PKA、PKC、MEK、tyrosine kinase、CaMII抑制劑處理下,皆能有效的抑制NO釋放與iNOS表現。Cycloheximide也能抑制cAMP的誘導作用,表示在NO與iNOS產生的過程,有新蛋白質合成。NF-κB抑制劑無法抑制cAMP誘導的NO,另外cAMP也不能刺激IκB-α分解、磷酸化與p65的轉移,由此推測cAMP誘導的NO釋放與iNOS表現的過程,並無NF-κB的參與。此外,cAMP能濃度相關的抑制LPS誘導的TNF-α釋放和TNF-α mRNA合成 , 所以cAMP的作用是經由抑制轉錄而來。然而cAMP卻無法抑制LPS誘發的IκB-α分解、磷酸化與p65的轉移,結果顯示cAMP抑制LPS誘導的TNF-α釋放,並不是經由抑制NF-κB的轉移而來。
綜合以上結果,(1) cAMP單獨處理下可經由PKA/pCREB誘發NO釋放與iNOS表現,而非NF-κB。(2) PDE4在調節cAMP誘導的功能上,扮演一個重要的角色。(3) cAMP抑制LPS誘導的TNF-α釋放,並不是經由抑制PKA的活化或NF-κB的轉移而來。

Alveolar macrophage (AM) are strategically located to function as a primary defense of the lung inhaled particulate matter, microorganisms, and environmental toxin. Using cultured rat alveolar NR8383 macrophages and primary alveolar macrophages (PAM), this study investigated the effect of cAMP and phosphodiesterase (PDE) on the production of nitric oxide (NO) and release of tumor necrosis factor-α (TNF-α). The cAMP analogues, dbt-cAMP and 8br-cAMP, induced NO formation and iNOS protein expression in a concentration- and time-dependent fashion. PDE activities were suppressed by both PDE3 and PDE4 inhibitors, but inhibition of PDE4 had a greater effect on potentiation of PGE1- and forskolin-induced NO formation and iNOS expression. This effect was consistent in the potentiation of cAMP level and phospho-cAMP response element binding protein (pCREB). Moreover, PKA inhibitor attenuated cAMP-induced NO production and pCREB expression. These results suggested that while basal level of cAMP are maintained, inhibitor of PDE4 may be more effective to increase the NO formation than those of PDE3 in alveolar macrophages. Treatment of cells with PKC, MEK, tyrosine kinase and CaMII inhibitor, all these inhibitors inhibited the cAMP-induced NO formation and iNOS expression. Cycloheximide prevented the cAMP-induced NO formation and iNOS protein expression, implying that new protein synthesis was required. NF-kB inhibitor failed to suppress cAMP-induced NO formation, and cAMP did not stimulated IkB-α degradation, IkB-α phosphorylation and p65 translocation, suggesting activation of NF-κB did not involve the cAMP-induced NO production and iNOS expression. Additionally, cAMP produced a concentration-dependent inhibition of TNF-α generation by LPS. Similar results, cAMP also induced a concentration-dependent inhibition of TNF-α mRNA expression, suggesting the inhibition of transcriptional process. However, cAMP failed to inhibit LPS-induced IkB-α degradation, IkB-α phosphorylation, and p65 translocation, indicating cAMP-induced inhibition of TNF-α release did not through the translocation of NF-κB.
In summary, these results indicate that in rat alveolar macrophages, (1) cAMP alone increases the production of NO and expression of iNOS through a PKA/pCREB, but not NF-κB, dependent pathway (2) PDE4, but not PDE3, plays a significant role on the regulation of cAMP-induced functions, and (3) inhibition of TNF-α release in LPS-activated macrophages by cAMP did not involve activation of PKA and translocation of NF-κB.

指導教授推薦書----------------------
論文口試委員會審書------------------
授權書------------------------------iii
誌謝--------------------------------iv
目錄--------------------------------v
縮寫表------------------------------vi
拮抗劑一覽表------------------------viii
中文摘要----------------------------ix
英文摘要----------------------------xi
研究動機與目的----------------------1
緒論--------------------------------3
實驗材料與方法----------------------14
結果--------------------------------24
討論--------------------------------32
圖表--------------------------------37
參考文獻----------------------------79

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