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研究生:徐佳如
研究生(外文):Chia-Ju Shu
論文名稱:BAY41-2272透過活化p38MAPK與JNK造成人類嗜中性白血球elastase釋放作用之探討
論文名稱(外文):BAY 41-2272 increases elastase release via p38 MAPK and JNK pathways in human neutrophils
指導教授:黃聰龍黃聰龍引用關係
指導教授(外文):T. L. Hwang
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:2008
畢業學年度:96
論文頁數:83
中文關鍵詞:嗜中性白血球
外文關鍵詞:neutrophils
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嗜中性白血球為宿主抵禦微生物入侵的首要防衛並且與發炎過程具有相關性。在嗜中性白血球中早已被證實有 soluble guanylyl cyclase (sGC) 及 protein kinase G (PKG) 的存在。儘管如此,guanosine 3’,5’-cyclic monophosphate (cGMP) 對於調節嗜中性白血球顆粒分泌 (granule secretion) 功能上的影響至今還是有爭論。另外,mitogen-activated protein kinases (MAPKs) 在調節人類嗜中性白血球,包含去顆粒化作用 (degranulation)、化學趨化作用 (chemotaxis) 以及呼吸爆破 (respiratory burst) 等生理功能上扮演著重要的角色。然而,對於 cGMP 與 MAPKs 訊號路徑之間的相關性目前仍然是未知。經研究證實,BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine) 是一個不經由 nitric oxide (NO) 的強效 sGC刺激劑,並能使 sGC 協同 NO 的作用。因此,本論文研究目的在於瞭解 BAY 41-2272 在人類嗜中性白血球調控去顆粒化作用上所扮演的角色及其作用機轉。實驗結果顯示,BAY 41-2272 不僅可以增加細胞內 cGMP 的含量,而且會大幅度地增強 sodium nitroprusside (SNP) 與 S-nitroso-N-acetyl-1,1-penicillamine (SNAP) 促進 cGMP的形成,而這些作用可被 sGC 抑制劑 ODQ 所抑制。BAY 41-2272 呈現濃度相關性地促進 p38 MAPK 和 JNK 活化以及提升 formyl-methionyl-leucyl-phen
ylalanine (fMLP) 誘導的 elastase 釋放作用。然而,sGC 抑制劑和 PKG 的拮抗劑不能抑制 BAY 41-2272 活化 p38 MAPK 與 JNK 的作用及 elastase 的釋出,並且 SNP 和 SNAP 也沒有加強 BAY 41-2272 的作用。這些實驗結果顯示 BAY 41-2272 活化 MAPK 的表現與 elastase 的釋放作用和 cGMP 訊號路徑無關。結果相同的與以一個可穿透細胞膜的 cGMP 類似物 8-Br-cGMP,並不影響 MAPKs 的磷酸化以及 elastase 之釋放。再者,BAY 41-2272具有濃度相關性的使mitogen-activated protein kinase kinase (MKK) 4 磷酸化,而非 MKK1/2 和MKK3/6,SNP 亦無法加強此活化作用。此外,p38 MAPK 與 JNK 的抑制劑可抑制 BAY 41-2272 加強 fMLP 活化嗜中性白血球造成的elastase 釋放反應。總結上述,在人類嗜中性白血球中,BAY 41-2272 是透過 cGMP 不相關,MKK4 相關的訊號路徑來活化 p38 MAPK 與 JNK 以及影響 elastase 的釋放作用。
Neutrophils are important in host’s defenses against invasion by microorganisms and are extensively involved in inflammatory processes. It has been reported that neutrophils contain soluble guanylyl cyclase (sGC) and protein kinase G (PKG). Despite this fact, the precise function of guanosine 3’,5’-cyclic monophosphate (cGMP) in regulating neutrophil granule secretion is still controversial. In addition, mitogen-activated protein kinases (MAPKs) play an important role in regulating human neutrophil functions, including degranulation, chemotaxis, and respiratory burst. However, the relationship between cGMP and MAPK signaling pathways is not well understood. BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,
4-b]pyridin-3-yl]-pyrimidin-4-ylamine) is a potent sGC stimulator in an nitric oxide (NO)-independent manner which stimulates the enzyme synergistically with NO. The aim of this study was to investigate the role of BAY 41-2272 on human neutrophil degranulation and its underlying mechanism. BAY 41-2272 not only increased cGMP levels but also greatly potentiated sodium nitroprusside (SNP)- and S-nitroso-N-acetyl
-1,1-penicillamine (SNAP)-induced cGMP formations, which was inhibited by the sGC inhibitor ODQ. BAY 41-2272 concentration-dependently promoted phosphorylation of p38 MAPK and JNK, and elevated formyl-methionyl-leucyl-phen
ylalanine (fMLP)-induced elastase release. However, p38 MAPK and JNK activations and elastase release caused by BAY 41-2272 were not inhibited by inhibitors of sGC and PKG, and enhanced by SNP and SNAP. These results suggested that cGMP pathway did not involve in BAY 41-2272 induced MAPK activations and elastase release. Consistent with this, the cell permeable cGMP analogue, 8-Br-cGMP, failed to induce phosphorylation of MAPKs and elastase release. In addition, BAY 41-2272 induced phosphorylation of mitogen-activated protein kinase kinase (MKK) 4, but not MKK1/2 and MKK3/6, in a concentration-dependent manner, which was not enhanced by SNP. Furthermore, BAY 41-2272 increased fMLP-induced elastase release was inhibited by inhibitors of p38 MAPK and JNK. In summary, these results demonstrate that BAY 41-2272 promoted p38 MAPK and JNK activation and elastase release via a cGMP-independent but MKK4-dependent signaling pathway in human neutrophils.
指導教授推薦書…………………………………………………
口試委員審定書…………………………………………………
論文授權書……………………………………………………III
誌謝……………………………………………………………IV
目錄…………………………………………………………… V
縮寫表…………………………………………………….1
中文摘要……………………………………………….....3
英文摘要……………………………………………….....4
研究動機………………………………………………….5
第ㄧ章、緒論……………………………….………………….7
第二章、實驗材料與方法…………………………………….27
第三章、實驗結果…………………………………………….31
第四章、討論………………………………………………….37
第五章、結論……………………………………………………46
附圖…………………………………………………………….47
參考文獻……………………………………………………….64
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