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

发布时间：2018-04-01 17:19

本文选题：巨噬细胞移动抑制因子　切入点：小干扰RNA　出处：《中国病理生理杂志》2013年10期

【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。
[Abstract]:Aim: to study the effect of small interference RNA-siRNAs on the inhibition of lipid inflammatory mediators release by glucocorticoid and its intracellular mechanism by blocking macrophage migration-inhibitory factor-MIF gene expression. Methods: mouse macrophage cell line RAW264.7 was cultured in vitro. The transfection efficiency of siRNA and the expression of MIF mRNA were detected by RT-PCR. The expression of MIF protein in RAW264.7 cells was detected by Western blotting. The anti-inflammatory effect of dexamethasone on MIF siRNA was observed after transfection of MIF siRNA. The content of prostaglandin E _ 2 (PGE _ 2) and leukotriene B _ 4 (LTB _ 4) in supernatant was detected by ELISA and the protein expression of Annexin _ 1 and A _ 2 伪 -C _ (PLA2 _ 伪) were detected by Western blotting. Results: compared with the negative control, ELISA siRNA could effectively block the fine cells. Expression of intracellular MIF protein, The effect of Dex on the expression of PGE2 and LTB4, the expression of cytoplasmic protein Annexin 1, and the phosphorylation of cPLA2 伪 in RAW264.7 cells were significantly enhanced. Conclusion the effects of glucocorticoid on the release of PGE2 and LTB4 were enhanced by glucocorticoid, and the release of PGE2 and LTB4 were inhibited by glucocorticoid. Blocking the expression of endogenous MIF protein could significantly enhance the sensitivity of RAW264.7 cells to glucocorticoid anti-inflammatory effects.
【作者单位】：第二军医大学附属长海医院烧伤科;
【基金】：国家自然科学基金资助项目(No.81000825)
【分类号】：R363

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