RNA干扰技术阻断小鼠巨噬细胞受体相互作用蛋白2表达的实验研究

目的观察阻断受体相互作用蛋白2（Rip2）对巨噬细胞产生炎症细胞因子的影响,以及对内毒素血症小鼠的保护作用。方法构建Rip2小干扰RNA（siRNh）重组表达质粒,转染细胞后RT．PCR和Western blot检测Rip2的mRNA和蛋白表达,四甲基偶氮唑盐（M1Tr）法检测细胞增殖水平,脂多糖（LPS）刺激后,测定TNFa和高迁移率组蛋白1（HMGB1）的水平。Rip2 siRNA质粒转染小鼠后,观察小鼠病死率,测定血清TNFct水平和肝组织Rip2和HMGB1表达。结果Rip2 siRNA表达质粒可阻断Rip2 mRNA和蛋白表达。Rip2阻断的细胞增殖明显,LPS刺激后产生TNFα、HMGB1减少;Rip2阻断的小鼠生存率较其他组高（P〈0．05）,肝组织中HMGB1[（40．21±11．03）Pg／g]表达和血清TNFα[（300．43±59．26）ng／L]水平均较其他组低（P〈0．05）。结论Rip2 siRNA表达质粒可阻断Rip2的表达,从而减少TNFα、HMGB1等炎症细胞因子的产生,降低小鼠内毒素血症的病死率。

A research on blockage of receptor-interacting protein 2 expression by small interfering RNA in murine macrophages

OBJECTIVE: To provide evidence that blocking the receptor-interacting protein 2 (Rip2) expression can decrease inflammatory cytokine production by macrophage and protect mice from endotoxin lethality. METHODS: Murine Rip2 small interfering RNA (siRNA) plamids were constructed and transfected into macrophages and Rip2 expression was assessed with RT-PCR and Western blot. Cell proliferation was assayed with MTT; TNFalpha concentration was assayed with ELISA and high-mobility group box 1 protein (HMGB1) level with semi quantitative Western blot after lipopolysaccharide (LPS) stimulation. LPS challenge was given after the plasmids were injected into mice and the survival rate was calculated. Rip2 and HMGB1 expression in liver was assessed with Western blot and serum TNFalpha level with ELISA. RESULTS: Rip2 siRNA plasmids could block the mRNA and protein expression of Rip2 and promote cell proliferation. Blocking of Rip2 could attenuate LPS-induced TNFalpha and HMGB1 production. HMGB1 expression in liver were decreased to (40.21 +/- 11.03) pg/g and serum TNFalpha level was decreased to (300.43 +/- 59.26) ng/L (P < 0.05). The survival rate of endotoxemic mice was also improved (P < 0.05). CONCLUSION: The results demonstrate that Rip2 siRNA plasmids can block the expression of Rip2, and decrease the production of TNFalpha and HMGB1, thus protect mice from lethal endotoxemia.