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目的探讨Cp G-c41分子对TLR9信号通路的干扰作用及其机制。方法获取小鼠骨髓巨噬细胞,M-CSF刺激使骨髓细胞向单核巨噬细胞分化,流式细胞术(flow cytometry,FCM)检测诱导成功的巨噬细胞的比例;培养Raw264.7细胞,ELISA法检测Cp G-c41分子对TLR9激动剂Cp G-1826刺激小鼠来源骨髓巨噬细胞和Raw264.7细胞诱发的炎症因子TNF-α和IL-6的影响;Western blot检测Cp G-c41对TLR9-My D88依赖型信号通路中磷酸化NF-κB蛋白表达的影响;ELISA法检测非Cp G-ODN分子对Cp G-1826刺激Raw264.7细胞诱发的炎症因子TNF-α和IL-6的影响;免疫荧光检测Cp G-c41与TLR9的共定位情况以及Cp G-c41对Cp G-1826与TLR9结合的影响。结果 Cp G-c41抑制Cp G-1826诱导的炎症因子TNF-α和IL-6的释放(P<0.01);Cp G-c41抑制TLR9信号通路中NF-κB的磷酸化表达;非Cp G-ODN不影响Cp G-1826诱导TLR9释放TNF-α和IL-6;Cp G-c41通过与TLR9竞争性结合抑制Cp G-1826与TLR9识别。结论 Cp G-c41通过竞争性结合TLR9干扰TLR9-My D88信号通路的活化。
Objective To investigate the interference effect of Cp G-c41 on TLR9 signaling pathway and its mechanism. Methods Mice bone marrow macrophages were obtained. M-CSF stimulated bone marrow cells to differentiate into mononuclear macrophages. Flow cytometry (FCM) was used to detect the percentage of macrophages that were induced successfully. Raw264.7 cells were cultured, The effect of CpG-c41 on TNF-α and IL-6 induced by stimulation of TLR9 agonist Cp G-1826 in mouse bone marrow-derived macrophages and Raw264.7 cells was detected by ELISA. Western blot was used to detect the expression of CpG-c41 CpG-1826 stimulation of Raw264.7 cells induced by inflammatory cytokines TNF-α and IL-6 (P <0.05) .Conclusion: CpG-1826 can stimulate the expression of phosphorylated NF-κB in TLR9-My D88-dependent signaling pathway. The co-localization of CpG-c41 and TLR9 was detected by immunofluorescence and the effect of Cp G-c41 on the binding of CpG-1826 to TLR9. Results Cp G-c41 inhibited the release of inflammatory cytokines TNF-α and IL-6 induced by CpG-1826 (P <0.01); CpG-c41 inhibited the phosphorylation of NF-κB in TLR9 signaling pathway; ODN did not affect CpG-1826-induced TLR9 release of TNF-α and IL-6; CpG-c41 inhibited CpG-1826 and TLR9 recognition by competitive binding to TLR9. Conclusion Cp G-c41 interferes with the activation of TLR9-My D88 signaling pathway by binding to TLR9.