论文部分内容阅读
目的 检测NLRC4在败血症儿童血液中的表达,并研究NLRC4在细胞因子产生中的重要性,以及脂多糖(LPS)诱导巨噬细胞中NLRC4表达的信号传导通路,并研究LPS对巨噬细胞中NLRC4表达的影响.方法 1.采用逆转录-定量聚合酶链反应和Western blot分析法,测定败血症组(n=42)和对照组(n=40)儿童NLRC4的mRNA和蛋白质表达水平;2.在体外使用LPS刺激RAW264.7巨噬细胞,并测定NLRC4及细胞因子表达水平,以研究LPS对NLRC4表达的调节作用;3.在LPS诱导下使用NLRC4 siRNA转染RAW264.7细胞,并再次测定NLRC4及细胞因子表达水平,研究NLRC4对产生炎症细胞因子的影响;4.阻断MAPK信号传导通路,测定NLRC4的表达水平.结果 1.与对照组相比,败血症组NLRC4的mRNA及蛋白质表达水平显著增加;2.NLRC4表达水平随着细胞使用含LPS的培养基培养时间的延长及浓度的增高而显著增加;3.在LPS诱导下经NLRC4 siRNA转染后的RAW264.7细胞中,IL-1β和IL-18的产生明显增加;4.阻断MAPK信号传导通路后,NLRC4的表达水平显著降低.结论 NLRC4在败血症儿童血清中的表达增加;Western blot的结果表明在RAW264.7细胞中LPS以时间和剂量依赖性方式诱导NLRC4表达;ELISA结果显示,通过NLRC4 siRNA转染RAW264.7细胞增强了LPS对IL-1β和IL-18产生的作用;LPS诱导NLRC4的表达水平与MAPK信号传导通路有关.“,”Objective To determine the expression of NLRC4 in the blood samples of children with septicaemia, and to investigate the importance of NLRC4 in cytokine production, lipopolysaccharide (LPS) inducing signal transduction pathway of NLRC4 expression in macrophages and the effect of LPS on the expression of NLRC4 in macrophages. Methods 1.The mRNA and protein expression levels of NLRC4 in 42 children with septicaemia and 40 normal controls were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. 2.LPS was used to stimulate RAW264.7 macrophages in vitro, and the expression of NLRC4 and cytokines were measured in order to study the regulatory effect of LPS on the expression of NLRC4. 3.In order to study the effect of NLRC4 on the production of inflammatory cytokines, NLRC4 siRNA was transfected into RAW264.7 cells under LPS induction and the expression levels of NLRC4 and cytokines were measured again. 4.The expression level of NLRC4 was measured after blocking the MAPK signaling pathway. Results 1.Compared with the control group, the expression of NLRC4 mRNA and protein in septicaemia group was significantly increased. 2.The expression level of NLRC4 was significantly increased with the prolongation of cell culture time and concentration of LPS-containing medium. 3.The production of IL-1β and IL-18 was significantly increased in RAW264.7 cells transfected with NLRC4 siRNA induced by LPS. 4.After blocking the MAPK signaling pathway, the expression level of NLRC4 was significantly decreased. Conclusion NLRC4 is more expressed in serum of sepsis children. The results from the Western blotting indicated that treatment with LPS induced NLRC4 expression in a time- and dose-dependent manner in RAW264.7 cells. A knockdown of NLRC4 by siRNA transfection enhanced the effect of LPS on IL-1β and IL-18 production, as determined by enzyme-linked immunosorbent assay. The expression of LPS-induced NLRC4 is associated with MAPK signaling pathway.