博碩士論文 972204016 詳細資訊




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姓名 林玉苑(Yu-Yuan Lin)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 巨噬細胞中抑制PDE4對LPS誘導發炎反應之調控
(Regulation of LPS-induced inflammatory responses by PDE4 inhibitor in macrophages)
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摘要(中) 第四型環狀核甘酸磷酸二酯酶 (PDE4) 為一可以水解第二傳訊者cAMP的酵素,因此有調節細胞中cAMP濃度與訊息傳遞的功能。在免疫細胞中,PDE的表現以PDE4為主,且實驗證實抑制PDE4活性有降低發炎反應的作用,故此類酵素被認為是免疫細胞功能的重要調節者。PDE4酵素家族包含四個基因,PDE4A、PDE4B、PDE4C與PDE4D。先前研究指出LPS刺激巨噬細胞可增加細胞內PDE4B的表現與活性,並釋放大量的TNF-?,而PDE4抑制劑rolipram會顯著抑制TNF-?的產生。在LPS活化TLR4同時也會增加細胞中Src tyrosine kinase的活性,給予Src tyrosine kinase抑制劑PP2亦可降低LPS誘導TNF-?的釋放。本研究結果顯示,在Raw 264.7細胞中,處理PP2會使LPS誘導TNF-?的釋放降低約50%,而rolipram與db-cAMP則幾乎可完全抑制此TNF-?的反應;在PDE4B基因剔除鼠的巨噬細胞中,LPS刺激TNF-?的釋放量會有明顯的下降,其降低程度與rolipram或PP2抑制野生型小鼠巨噬細胞的程度相當,顯示PDE4B可能藉由調節cAMP與Src tyrosine kinase的訊息傳導路徑以調控LPS/TNF-?的反應。我們發現在Raw 264.7細胞中,PP2不會抑制LPS所誘導的PDE4活性,這結果排除了PP2抑制TNF-?的釋放可能是由於其抑制PDE4B活性的可能性。LPS刺激巨噬細胞會使細胞內ROS增加,而此反應可被cAMP與PKA所抑制,但是抑制Src tyrosine kinase活性却不會影響ROS的產生。在Raw 264.7細胞中,LPS對Lyn-tyr396的磷酸化沒有明顯影響,而Lyn-tyr507的磷酸化卻有抑制的現象,同時,rolipram對Lyn的磷酸化表現也與預期不符。綜合本研究結果我們發現,在巨噬細胞中,抑制PDE4B可以降低LPS刺激的TNF-?與ROS產生,而抑制Src tyrosine kinases只會降低TNF-?釋放不會影響ROS產生,此差異顯示cAMP訊息傳導與Src tyrosine kinases活化狀態在TLR訊息傳導中有其調控之專一性。
摘要(英) Type 4 cAMP-specific phosphodisterases (PDE4s), the enzymes that degrade the second messenger cAMP, play a critical role in regulation of intracellular cAMP concentration. It has been demonstrated that PDE4 isozymes are expressed at high levels in immune cells and PDE4 inhibitors exhibit anti-inflammatory effects. revious studies have shown that lipopolysaccharide (LPS) induced PDE4B expression and activity in macrophages, and this induction is associated with an increase in the production of TNF-?. This TNF-? responses was inhibited by the PDE4 inhibitor rolipram. Moreover, LPS stimulates an increase in src tyrosine kinase activity, while is involved in various function of macrophages. In this study, we observed that rolipram, db-cAMP, and the Src kinase inhibitor PP2 attenuate LPS-induced TNF-? production in Raw 264.7 cells. In PDE4B-deficient macrophages, LPS-induced TNF-? release was decreased and the level of the decrease was similar to that observed in the wild-type macrophages inhibited by rolipram or PP2, indicating the effects of PDE4B on the TNF-? release are mediated by regulating cAMP signaling pathway as well as src tyrosine kinase activity. Moreover, measurements of PDE enzymatic activity indicated that LPS-induced PDE4 activity in macrophages was not inhibited by PP2, ruling out the possibility that PP2 attenuated TNF-? response isn’t derived from the inhibition of PDE4 by PP2. We also found that LPS-induced ROS production in macrophages was inhibited by cAMP or PKA activation, but not by PP2. In Raw 264.7 cells, LPS did not alter Lyn-tyr396 phosphorylation while decreased Lyn-tyr507 phosphorylation. In addition, the effect of rolipram on Lyn tyrosine phosphorylation was not exhibited as predicted. Taken together, these findings showed that inhibition of PDE4B activity reduces LPS-induced TNF-? release and ROS production, but interupting Src tyrosine kinase only attenuates the TNF-? release. This difference indicates that cAMP signaling and Src tyrosine kinase activation have their unique and specific role in regulation of TLR signaling.
關鍵字(中) ★ 反應性含氧物種
★ 腫瘤壞死因子
★ Src 酪胺酸酶
★ 磷酸雙酯酶
★ 脂多糖
關鍵字(英) ★ LPS
★ PDE4
★ Src tyrosine kinase
★ TNF-a
★ ROS
論文目次 中文摘要 i
英文摘要 ii
目錄 iii
圖目錄 v
縮寫檢所表 vi
第一章 緒論 1
1.1環狀核苷酸磷酸二酯酶 (PDE) 1
1.2第四家族環狀核苷酸磷酸二酯酶 (Type 4 PDE;PDE4) 與巨噬細胞的免疫調控 2
1.3 Toll-like receptor (TLR) 之訊息傳導 3
1.4反應性含氧物種 (ROS) 4
1.5 ROS與免疫反應 5
1.6 Src tyrosine kinases 6
第二章 研究目的 9
第三章 實驗材料與方法 10
3.1實驗材料 10
3.2 實驗方法 11
3.2.1巨噬細胞之培養與處理方法 11
3.2.1.1 Raw 264.7細胞之培養與處理 11
3.2.1.2小鼠巨噬細胞收取培養與處理 12
3.2.1.3 HEK 293細胞之培養 13
3.2.2酵素免疫分析法 (ELISA) 14
3.2.3 PDE活性之檢測 14
3.2.4細胞萃取液之收集 15
3.2.5蛋白質濃度測定 16
3.2.6電擊轉染作用 (Electroporation transfection) 16
3.2.7 ROS含量測定 17
3.2.8西方墨點分析 17
第四章 實驗結果 20
4.1 LPS刺激巨噬細胞之TNF-?釋放會被cAMP與PP2抑制 20
4.2剔除PDE4B基因會降低LPS刺激巨噬細胞之TNF-?釋放 20
4.3 PP2對LPS誘導的PDE4活性無抑制作用 21
4.4 LPS誘導巨噬細胞產生ROS 22
4.5 PDE抑制劑對LPS刺激巨噬細胞產生ROS的影響 22
4.6 Src tyrosine kinase對LPS誘導巨噬細胞產生ROS之影響 23
4.7 Rolipram對Src與Lyn tyrosine磷酸化之影響 23
第五章 討論 25
5.1巨噬細胞中LPS誘導TNF-?表現與Src tyrosine kinase之關係 25
5.2 Src tyrosine kinase與PDE調控訊息傳遞之關係 26
5.3 LPS誘導細胞表現ROS與PDE調控cAMP之訊息傳遞 28
5.4巨噬細胞中Src tyrosine kinase與ROS表現之關係 29
第六章 參考文獻 30
圖 36
附錄一 46
附錄二 47
附錄三 48
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指導教授 金秀蓮(S.-L. Catherine Jin) 審核日期 2010-8-24
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