AIM: To investigate the effects of chemically synthesized small interfering RNA ( siRNA ) targeting to NF - κB p65 expression on the production of inflammatory mediators in lipopolysaccharide ( LPS ) - activated mouse macrophage Ana - 1 cells.METHODS: The siRNA targeting to mouse NF - κB p65 mRNA was transfected into Ana - 1 cells and the rates of transcription and translation of NF - κB p65 were detected by RT - PCR and Western blotting 24 h after transfection.The cytokines ( such as TNF -α, IL - 1β, IL-6 and IL - 10 ) in the supernatant of Ana - 1 cells activated by LPS ( 1 mg/L ) were measured by ELISA at 0 h, 4 h, 12 h and 24 h after transfection.RESULTS: The expression of the NF - κB p65 at mRNA and protein levels in Ana - 1 cells was specifically and extensively suppressed by siRNA 24 h after transfection ( P ＜ 0.01 ).The levels of TNF - α, IL - 1β and IL - 6 in the supernatant of Ana - 1 cells were cut down after transfection with NF - κB p65 siRNA in the interfering group ( P ＜0.05 ).In contrast, the level of IL - 10 was increased, especially at 12 h and 24 h ( P ＜0.05 ).CONCLUSION: The technique of RNAi effectively silences the expression of NF - κB p65 gene in mouse macrophage Ana - 1 cells, thus causes the decrease in the production of pro - inflammatory cytokines and the increase in IL - 10 expression.%目的:探讨RNA干扰(RNAi)技术在抑制NF-κB p65表达、调控LPS活化后巨噬细胞细胞因子表达中的应用.方法:利用阳离子脂质体将NF-κB p65小干扰RNA(siRNA)瞬时转染入Ana-1细胞,RT-PCR及Western blotting法检测其沉默效率,ELISA法检测LPS(1 mg/L)刺激下0 h、4 h、12 h和24 h Ana-1细胞培养上清中TNF-α、IL-1β、IL-6和IL-10浓度.结果:NF-κB p65 siRNA转染24 h后,NF-κB p65在基因水平及蛋白水平表达均被明显抑制(P<0.05).RNA干扰组Ana-1细胞培养上清中细胞因子TNF-α、IL-1β和IL-6表达在相应时点内均较对照组明显降低(P<0.05),IL-10表达显著升高,在12 h和24 h差异显著(P<0.05).结论:体外实验初步证实RNAi技术能有效沉默小鼠巨噬细胞NF-κB p65基因的表达,下调其下游调控的促炎症细胞因子TNF-α、IL-1β、IL-6及上调抑炎症细胞因子IL-10的表达,从而抑制过度的炎症反应.
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