本文選題：巨噬細(xì)胞移動抑制因子　切入點：小干擾RNA　出處：《中國病理生理雜志》2013年10期

【摘要】：目的:研究小干擾RNA(siRNA)阻斷巨噬細(xì)胞移動抑制因子(macrophage migration-inhibitory factor,MIF)基因表達(dá)對糖皮質(zhì)激素抑制脂質(zhì)炎癥介質(zhì)釋放的影響及其細(xì)胞內(nèi)機制。方法:體外培養(yǎng)小鼠巨噬細(xì)胞系RAW264.7,采用免疫熒光法觀測siRNA轉(zhuǎn)染效率,RT-PCR檢測MIF mRNA的表達(dá),Western blotting檢測MIF蛋白的表達(dá);RAW264.7細(xì)胞轉(zhuǎn)染MIF siRNA后觀察地塞米松(Dex)抗炎作用的變化,用ELISA檢測細(xì)胞上清中前列腺素E2(PGE2)和白三烯B4(LTB4)的含量,Western blotting檢測胞漿膜聯(lián)蛋白Annexin 1和下游胞漿磷酸酯酶A2α(cPLA2α)的蛋白表達(dá)變化。結(jié)果:與陰性對照相比,MIF siRNA能有效阻斷細(xì)胞內(nèi)源性MIF蛋白的表達(dá),增強RAW264.7細(xì)胞對Dex作用的敏感性;明顯增強Dex抑制PGE2和LTB4產(chǎn)生的效應(yīng),增加胞漿蛋白Annexin 1的表達(dá),抑制cPLA2α的磷酸化。結(jié)論:MIF siRNA能增強糖皮質(zhì)激素抑制脂質(zhì)炎癥介質(zhì)PGE2和LTB4的釋放,且可能是通過影響Annexin 1-cPLA2α信號通路實現(xiàn)的。阻斷內(nèi)源性MIF蛋白的表達(dá)可顯著增強RAW264.7細(xì)胞對糖皮質(zhì)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 偽 -C _ (PLA2 _ 偽) 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 偽 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.
【作者單位】： 第二軍醫(yī)大學(xué)附屬長海醫(yī)院燒傷科;
【基金】：國家自然科學(xué)基金資助項目(No.81000825)
【分類號】：R363

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