iNOS與Src於LiCl誘發巨噬細胞移行之相關探討

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題名:	iNOS與Src於LiCl誘發巨噬細胞移行之相關探討 The iNOS/Src axis contributes to LiCl-mediated macrophage migration
作者:	簡婉竹
貢獻者:	基礎醫學研究所碩士班
關鍵詞:	LiCl，Src，iNOS，巨噬細胞移行能力 LiCl, Src, iNOS, macrophage migration
日期:	2011-07-27
上傳時間:	2011-10-17 16:53:25 (UTC+8)
出版者:	中國醫藥大學
摘要:	Wnt 訊息傳遞參與在許多的發炎反應。儘管如此，它的免疫調控生物功能還是持續被研究中，由於巨噬細胞在先天性免疫反應扮演重要的角色，因此我們想知道Wnt訊息傳遞是否有可能參與在巨噬細胞的活化。lithium chloride (LiCl)是glycogen synthase kinase (GSK3β)的抑制劑，進而可以活化Wnt訊息傳遞。因此我們觀察LiCl 有無影響巨噬細胞移行的能力，結果我們發現，無論RAW264.7巨噬細胞或老鼠的peritoneal macrophages (PEMs)，其在LiCl刺激之後，細胞移行能力與整體蛋白tyrosine phosphorylation表現均明顯增加。若細胞以PP2 (Src family kinases〔SFKs〕抑制劑)進行前處理，則LiCl誘發的移行能力會顯著下降。因此我們認為Src family kinases有可能參與在此過程。有趣的是，LiCl會增加Src的表現量，但Lyn、Fgr、Hck 的蛋白量卻不受 LiCl的刺激而有所改變；更進一步，利用siRNA的技術，將Src的表現量減少，同時也會減少LiCl刺激的巨噬細胞移行與FAK Pi-Tyr861；但如果回復Src的表現量，則可以恢復LiCl刺激細胞的移行能力。此外，其他GSK3β抑制劑 (例如: SB216763 and kenpaullone)刺激RAW264.7巨噬細胞後，同LiCl般，也會增加Src和FAK Pi-Tyr861的表現量與細胞移行能力。特別的是，我們也發現了在缺乏iNOS的巨噬細胞，其LiCl所誘發Src的表現，活性與細胞移行能力皆減弱。由此可知，LiCl誘增Src與細胞的移行是需要iNOS參與。從上述結果，我們總結LiCl刺激後，GSK3β活性的下降，會造成iNOS的增加，進而提高Src的表現量以及Src與FAK的活性，加速巨噬細胞的移行。 The Wnt signaling has been implicated in certain inflammatory responses; however its biological role in the inflammatory regulation remains to be characterized. Due to macrophages are critical in innate immunity, thereby we wonder whether Wnt signaling might be involved in their activation. Considered lithium chloride (LiCl) is an inhibitor of glycogen synthase kinase3β (GSK3β) whose activation represses Wnt signaling; therefore we first checked whether LiCl could affect macrophage migration. We observed that upon LiCl stimulation, the motility and the content of total protein tyrosine phosphorylation in RAW264.7 macrophages and mice peritoneal macrophages (PEMs) were significantly increased. The inhibition of LiCl-induced macrophage migration by PP2 (an inhibitor for Src family kinases (SFKs)) suggested the involvement of SFKs in this process. Interestingly, while Src was greatly induced, the expression of its myeloid relatives (i.e. Lyn, Fgr, Hck) was almost unaltered in RAW264.7 cells exposed to LiCl. Attenuation of Src suppressed LiCl-elicited movement and the level of FAK Pi-Tyr861, which could be reversed by the reintroduction of siRNA-resistant Src. Similar increment of Src, FAK Pi-Tyr861 and migration ability could also be detected in RAW264.7 treated with other GSK3β inhibitors (i.e. SB216763 and kenpaullone). Consistent with Src and migration increment was iNOS-dependent in macrophages, markedly reduced Src expression, activity and cell migration was observed in iNOS-null PEMs. Based on these results, we concluded that through inhibition of GSK3β, LiCl caused induction of iNOS, upregulation of the expression and activity of Src, and FAK activation that ultimately led to macrophage movement.
顯示於類別:	[基礎醫學研究所] 博碩士論文

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