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目的:探讨牙龈卟啉单胞菌毒力因子环二鸟苷酸(c-di-GMP)对M1型巨噬细胞极化的调控作用及其体外促炎作用。方法:使用不同质量浓度(0.25、1、4、16、64μg/m L)的c-di-GMP分别处理RAW 264.7细胞3、6、12、24 h,采用实时定量RTPCR、ELISA及流式细胞术检测炎症因子肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-6以及巨噬细胞M1型极化标志细胞因子IL-23和M2型极化标志细胞因子IL-10的mRNA、蛋白质和细胞表面标志物的表达。结果:RAW 264.7细胞TNF-α、IL-6和IL-23的mRNA表达水平随c-di-GMP的浓度和时间的变化均有不同程度的增高,具有剂量和时间依赖性,而IL-10的表达水平在各组中无显著升高;ELISA结果与实时定量RT-PCR结果基本相同;流式细胞术结果显示在c-di-GMP刺激RAW 264.7细胞24h后,细胞表面分子F4/80与CD86双阳性率、CD86单阳性率明显增高。结论:c-di-GMP能够刺激M1型巨噬细胞相关细胞因子IL-23及炎症因子TNF-α和IL-6的表达,诱导巨噬细胞发生M1型巨噬细胞极化,进而导致炎症反应,提示c-di-GMP可能通过诱导巨噬细胞发生M1型极化,进而在牙周炎的发生、发展过程中发挥重要作用。
Objective: To investigate the regulation effect of c-di-GMP against Porphyromonas gingivalis on the polarization of M1 macrophages and its proinflammatory effects in vitro. Methods: RAW 264.7 cells were treated with c-di-GMP at different concentrations (0.25, 1, 4, 16 and 64μg / mL for 3, 6, 12 and 24 h respectively). Real-time quantitative RTPCR, ELISA and flow cytometry (TNF-α), interleukin (IL) -6, macrophage M1-type marker IL-23 and M2-type IL-10 mRNA were detected. Expression of proteins and cell surface markers. Results: The mRNA expression levels of TNF-α, IL-6 and IL-23 in RAW 264.7 cells were increased in a dose-and time-dependent manner with the concentration and time of c-di-GMP, The results of flow cytometry showed that after c-di-GMP stimulation of RAW 264.7 cells for 24h, the expression of F4 / 80 CD86 double positive rate, CD86 single positive rate was significantly higher. Conclusions: c-di-GMP can stimulate the expression of M1-related macrophages IL-23 and inflammatory cytokines TNF-α and IL-6 and induce the macrophage’s polarization of M1 macrophages, leading to inflammation , Suggesting that c-di-GMP may play an important role in the occurrence and development of periodontitis by inducing M1-type polarization in macrophages.