siRNA沉默MIF基因对糖皮质激素抑制脂质炎症介质释放的影响及其机制  

Influence of siRNA-mediated MIF knockdown on inhibition of inflammatory lipid mediator release by glucocorticoids

作　　者：孙瑜[1] 韩姝 王俊杰[1] 夏照帆[1] 

机构地区：[1]第二军医大学附属长海医院烧伤科,上海200433

出　　处：《中国病理生理杂志》2013年第10期1803-1808,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81000825)

摘　　要：目的:研究小干扰RNA(siRNA)阻断巨噬细胞移动抑制因子(macrophage migration-inhibitory factor,MIF)基因表达对糖皮质激素抑制脂质炎症介质释放的影响及其细胞内机制。方法:体外培养小鼠巨噬细胞系RAW264.7,采用免疫荧光法观测siRNA转染效率,RT-PCR检测MIF mRNA的表达,Western blotting检测MIF蛋白的表达;RAW264.7细胞转染MIF siRNA后观察地塞米松(Dex)抗炎作用的变化,用ELISA检测细胞上清中前列腺素E2(PGE2)和白三烯B4(LTB4)的含量,Western blotting检测胞浆膜联蛋白Annexin 1和下游胞浆磷酸酯酶A2α(cPLA2α)的蛋白表达变化。结果:与阴性对照相比,MIF siRNA能有效阻断细胞内源性MIF蛋白的表达,增强RAW264.7细胞对Dex作用的敏感性;明显增强Dex抑制PGE2和LTB4产生的效应,增加胞浆蛋白Annexin 1的表达,抑制cPLA2α的磷酸化。结论:MIF siRNA能增强糖皮质激素抑制脂质炎症介质PGE2和LTB4的释放,且可能是通过影响Annexin 1-cPLA2α信号通路实现的。阻断内源性MIF蛋白的表达可显著增强RAW264.7细胞对糖皮质激素抗炎作用的敏感性。AIM：To investigate the influence of siRNA-mediated macrophage migration inhibitory factor （MIF） knockdown on inhibition of inflammatory lipid mediator release by glucocorticoids. METHODS：Mouse macrophage cell line RAW2647 was transiently transfected with MIF siRNA and control siRNA by liposome method. The transfection efficiency was assessed by immunofluorescence technique. The expression of MIF mRNA and protein was examined by RT-PCR and Western blotting, respectively. Prostaglandin E2 （PGE2） and leukotriene B4 （LTB4） production in cell culture supernatants was measured by ELISA, and the protein expression of Annexin 1, cytosolic phospholipase A2α （cPLA2α） and phospho-cPLA2α were evaluated by Western blotting. RESULTS：MIF siRNA significantly inhibited MIF expression both at mRNA and protein levels in RAW2647 cells and subsequently enhanced the inhibitory effect of dexamethasone （Dex） on PGE2 and LTB4 production. MIF siRNA also increased Annexin 1 expression becreased by Dex, and strengthened the inhibitory effect of Dex on the phosphorylation of cPLA2α. CONCLUSION：MIF siRNA enhances the inhibitory effect of Dex on PGE2 and LTB4 production from RAW2647 cells partly via increasing Annexin 1 expression and inhibiting cPLA2α phosphorylation. Intracellular MIF knockdown mediated by siRNA may enhance the sensitivity of RAW2647 cells to the anti-inflammatory effect of glucocorticoids.

关 键 词：巨噬细胞移动抑制因子 小干扰RNA 糖皮质激素 脂质炎症介质 

分 类 号：R392.12[医药卫生—免疫学]