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【目的】探索猪链球菌2型感染后单核/巨噬细胞启动信号转导通路机制,探讨荚膜唾液酸对细菌激活巨噬细胞TLR2-AKT-NF-κB信号通路的影响。【方法】以小鼠巨噬细胞系RAW264.7细胞为研究对象,采用RT-PCR、Western blotting、免疫荧光和ELISA法分别检测猪链球菌2型野毒株、唾液酸缺失突变株、唾液酸回复突变株感染后不同时间点巨噬细胞TLR2 mRNA转录水平、AKT磷酸化水平、NF-κB激活程度以及前炎症因子TNF-α分泌水平;再分别用TLR2阻断剂和PI-3K抑制剂预处理巨噬细胞,检测上述分子的表达水平。【结果】唾液酸缺失株可选择性的活化信号转导通路途径。RT-PCR结果表明,缺失株TLR2 mRNA表达水平自1 h开始升高,1.5 h达高峰后有所下降;Westernblotting显示,缺失株TLR2蛋白表达水平7 h达高峰,9 h下降;p-AKT水平1.5 5 h持续稳定在高峰水平,7 h后开始下降;免疫荧光可见15 min NF-κB激活-核转运程度较高;ELISA结果显示,10 h之后TNF-α的水平显著高于野生株和回复株。使用TLR2阻断剂和PI-3K抑制剂,三菌株通路活化程度均明显受抑制。【结论】荚膜唾液酸可抑制宿主免疫细胞TLR2-AKT-NF-κB信号通路的激活,藉此参与细菌逃避宿主的免疫防御作用。
【Objective】 To explore the mechanism of monocyte / macrophage activation signal transduction pathway after Streptococcus suis type 2 infection and to explore the effect of capsular sialic acid on TLR2-AKT-NF-κB signaling pathway by macrophages. 【Method】 The murine macrophage cell line RAW264.7 cells were used as the research object. RT-PCR, Western blotting, immunofluorescence and ELISA were used to detect Streptococcus suis type 2 wild type, sialic acid deletion mutant, sialic acid The level of TLR2 mRNA transcription, the phosphorylation of AKT, the activation of NF-κB and the secretion of TNF-α of proinflammatory cytokines of macrophages were detected at different time points after infection with the recombinant plasmids. The TLR2 inhibitor and PI-3K inhibitor Macrophages were treated and the expression levels of these molecules tested. 【Result】 The sialic acid-deleted strains could selectively activate the signal transduction pathway. RT-PCR results showed that TLR2 mRNA expression level of deletional strain increased from 1 h and decreased after 1.5 h. Western blotting showed TLR2 protein expression peaked at 7 h and decreased at 9 h; p-AKT level After 1.5 h, the level of NF-κB activation and nuclear transport was higher at 15 min, and the level of TNF-α after 10 h was significantly higher than that of wild-type and response Strain. With TLR2 blockers and PI-3K inhibitors, the degree of activation of the three bacterial pathways was significantly inhibited. 【Conclusion】 Capsular sialic acid can inhibit the activation of TLR2-AKT-NF-κB signaling pathway in host immune cells, thereby participating in the immune evasion of the host.