RNA干扰对人肺泡上皮A549细胞巨噬细胞移动抑制因子基因表达抑制作用

目的探讨巨噬细胞移动抑制因子(MIF)小干扰RNA(siRNA)干扰对人肺泡上皮A549细胞株MIF基因表达的影响.方法将MIF siRNA用转染剂Interferin介导转染体外培养的A549细胞,通过RT-PCR方法观察不同浓度MIF siRNA转染后,对MIF mRNA表达的影响;采用细胞免疫荧光方法分析MIF蛋白表达的强弱;应用MTT方法测定MIF siRNA转染对细胞的毒性,及脂多糖(LPS)刺激对A549细胞生存率的影响.结果 (1)MIF siRNA转染人肺泡上皮A549细胞株后,可下调细胞中MIF mRNA的表达,只有当MIF siRNA转染浓度大于50 nmol/L时,才能明显下调MIF mRNA的表达;(2)MIF siRNA转染人肺泡上皮A549细胞株后,A549细胞MIF的荧光蛋白表达也明显降低,在此基础上再加入MIF刺激,MIF的蛋白表达又明显增加;(3)MIF siRNA转染对A549细胞几乎没有毒性,且对LPS刺激A549细胞导致细胞死亡及细胞的生存率下降有一定的保护作用.结论 MIF siRNA能在A549细胞特异沉默目的基因MIF和抑制MIF蛋白的表达,且对LPS刺激导致细胞死亡有一定的保护作用.

The gene silencing mediated by RNA interference on macrophage migration inhibitory factor expression in human alveolar epithelial cell line A549

Objective To study the inhibitory effect on macrophage migratory inhibitory factor (MIF) expression by MIF small interference RNA (siRNA)in human alveolar epithelial cell line A549. Methods After MIF siRNA was transfected into human alveolar epithial cells A549 with Interferin, RT-PCR was carried out to assess the dose-response of MIF siRNA's effect on MIF expression. Cell immumofluorescence method was used to analyze the expression of MIF protein. MTT cell proliferation assay was performed to detect the cytotoxicity of MIF siRNA to A549 cells, and the survival rate of A549 cells caused by lipopolysaccharide(LPS). Results (1) Only when the concentration was over 50 nmol/L, MIF siRNA significantly downregulated the MIF mRNA expression. (2) MIF siRNA also greatly reduced the expression of MIF fluorescent protein, which could be reversed by MIF. (3)MIF siRNA transfection had almost no toxicity to A549 cells, but also could reduce the cell mortality and increase the survival rate caused by LPS. Conclusions MIF siRNA can specially silence the expression of target gene MIF mRNA and reduce the MIF protein in A549 cells, therefore MIF siRNA may exert its safeguard to the death of cell induced by LPS.