【摘要】 目的:研究巨噬细胞移动抑制因子siRNA对脂多糖刺激的肺泡上皮细胞Toll样受体(TLR)及其下游信号转导通路的影响和作用机制。方法:用LPS刺激A549细胞,脂质体法分别将巨噬细胞移动抑制因子(MIF)siRNA和非特异性siRNA加入A549细胞进行干预。RT-PCR法检测MIF和TLR2、TLR4 mRNA的表达,Western Blot法分析TLR下游信号转导通路元件髓样分化因子88(MyD88)和干扰素调节因子3(IRF3)蛋白表达,细胞免疫荧光法观察NF-κB核迁移,ELISA法测定细胞培养上清炎症介质TNF-α、IL-1β、IL-6,免疫介质IFN-β分泌水平。结果:MIF siRNA对LPS刺激诱导的MIF和TLR2、TLR4mRNA高表达有显著的下调作用和部分阻断NF-κB(p65)核迁移。LPS刺激诱导后,MIF siRNA显著降低MyD88蛋白表达和炎症介质TNF-α、IL-1β和IL-6的分泌水平,但MIF siRNA对IRF3蛋白表达和免疫介质IFN-β分泌水平无影响。结论:MIF siRNA能抑制A549细胞的炎症介质TNF-α、IL-1β、IL-6过度分泌,其机制可能是MIF siRNA降低了LPS刺激A549细胞诱导的MIF和TLR2、TLR4高表达,及阻断TLR下游信号转导通路元件MyD88和NF-κB核迁移。更多还原

【Abstract】 AIM: To investigate the effect and mechanism of the small interfering RNAs(siRNA) for macrophage migration inhibitory factor(MIF) on expression of TLRs and downstream signaling components in alveolar epithial cell line A549 stimulated by lipopolysaccharide(LPS).METHODS: MIF siRNA and nonspecific siRNA were transfected into A549 cells by liposome respectively,then these cells were stimulated with LPS.The expression of MIF,TLR2,TLR4 mRNA were measured by RT-PCR.The proteins expression of MyD88 and IRF3 of TLR’s downstream signaling components were analyzed by Western Blot.The nuclear translocation of NF-κB was observed by cell immunofluorescence.The cells supernatant was collected and the inflammatory factors such as TNF-α,IL-1β,IL-6,IFN-β were measured by ELISA.RESULTS: LPS could stimulated the high expression of MIF,TLR2,TLR4 mRNA,and MIF siRNA could down regulate them and partially blocked NF-κB(p65) nuclear translocation.MIF siRNA also decreased significantly the high expression of MyD88 and the secretion levels of TNF-α,IL-1β,IL-6,but MIF siRNA did not work to IRF3 and IFN-β.CONCLUSION: MIF siRNA can inhibit the over-secretion of the inflammatory mediators TNF-α,IL-1β,IL-6 in A549 cells stimulated by LPS.The mechanism may be MIF siRNA reduced the highly expression of MIF,TLR2,and TLR4 mRNA,and blocked MyD88 protein of TLR downstream signal transduction pathway and the migration of NF-κB nuclear.更多还原