目的 通过小干扰RNA(siRNA)使小鼠腹腔巨噬细胞Tak1基因沉默,观察Tak1基因对腹腔巨噬细胞细胞因子释放的影响.方法 以靶向Tak1基因的siRNA作为实验组,同时设立空白对照组,瞬时转染昆明种小鼠腹腔巨噬细胞,采用荧光定量PCR技术定量检测Tak1 mRNA表达水平;采用Western blotting技术测定Tak1表达抑制情况,进而实现Tak1 siRNA干扰作用的优化;并观察LPS攻击后腹腔巨噬细胞IL-1β的表达水平.结果 细胞分别转染0.6、1.2、1.6 μmol/L Takl siRNA,mRNA抑制率为69.73%、80.85%、88.78%,最佳转染浓度为1.6μmol/L.Tak1蛋白在转染48h出现良好沉默效果,以1.6 μmol/L siRNA效果最好.转染后小鼠腹腔巨噬细胞在100 ng/ml LPS刺激下,细胞因子IL-1β分泌水平明显升高(P＜0.01).结论 靶向Tak1基因的siRNA具有较好的靶基因沉默效果.%Objective To investigate the biological effects of Tak1 siRNA on mouse peritoneal macrophage.Methods The Tak1 siRNA was used for instant transfection on the mouse peritoneal macrophage, the knockdown of Tak1 gene was evaluated by detecting Tak1 mRNA expression level with Q-RT-PCR, and the Tak1 protein expression level was detected by Western blotting technique.The interference of Tak1 siRNA on Tak1 gene was optimized, and the IL-1β releasing level was detected by ELISA.Results The Q-RT-PCR result showed the inhibitory effect was 69.73％, 80.85％, 88.78％ in the dose of 0.6, 1.2, 1.6 μmol/L Tak1 siRNA respectively, so 1.6 μmol/L dose was used in the formal test.In Western blotting assay, the expression of Tak1 protein was inhibited by Tak1 siRNA in the dose dependent manner.The LPS stimulated IL-1β releasing from the transfected macrophage showed that IL-1β level was elevated in Tak1 siRNA treated group (P ＜ 0.01).Conclusion The Tak1 siRNA can knock down Tak1 gene effectively in mouse peritoneal macrophage.
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