摘要目的 研究类风湿关节炎(RA)患者外周血单个核细胞(PBMCs) Toll样受体4(TLR4)通过诱导Th17细胞分化参与RA疾病发生发展的意义.方法 采集22例RA患者和20名健康对照外周血,流式细胞术检测Th17细胞频率,脂多糖刺激PBMCs 2 d,实时荧光定量聚合酶链反应(RT-qPCR)检测PBMCs TLR4 mRNA水平,酶联免疫吸附试验(ELISA)法检测上清液白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α的含量.收集上清液与CD4+脐带血单个核细胞(CBMCs)培养3d,RT-qPCR检测CBMCsIL-17 mRNA水平.ELISA检测上清液IL-17含量.统计学处理采用成组t检验.结果 与健康对照组比较,RA患者Th17细胞频率[(0.53±0.14)％与(1.57±0.68)％]、TLR4 mRNA显著升高(P均＜0.01)；PBMCs经脂多糖刺激后,RA患者TLR4 mRNA升高3.5倍,而健康对照组下降到0.11倍；RA患者PBMCs产生的IL-6、TNF-α较健康对照组明显增加(P＜0.01)；RA患者PBMCs上清液诱导CD4+CBMCs IL-17 mRNA水平、IL-17含量与健康对照组相比均显著升高(P＜0.01)；而未经脂多糖刺激,2组差异无统计学意义(P＞0.05).结论 RA患者PBMCs的TLR4表达及对脂多糖的刺激的反应性增加,且具有较强的诱导Th17细胞分化能力.

abstractsObjective To explore the effect of Toll-like receptor 4 (TLR4) on the peripheral blood mononuclear cells(PBMCs) in the development of rheumatoid arthritis (RA) by inducing the differentiation of Th17 cells.Methods Forty two subjects were recruited to the study,including 22 patients with RA and 20 healthy controls (HC).The percentage of circulating Th17 cells were analyzed using flow cytometry.PBMCs were stimulated with LPS for two days.TLR4 mRNA of the PBMCs and the concentration of IL-6 and TNF-α in the supernatants were analyzed with the real-time qPCR and ELISA respectively.Supematants was then used for CD4+ cord blood mononuclear cells (CBMCs) culture,and IL-17 in the supernatants and the expression of IL-17 mRNA in cells were detected by ELISA and real-time qPCR respectively.The statistical analysis was carried out with t-test.Results The TLR4 mRNA expression in the PBMCs and the percentage of circulating Th17 ceils of RA patients were significantly higher than that of the HC group (P＜0.01).Stimulated by LPS,the expression of TLR4 mRNA increased 3.5 times in the RA group but decreased 0.11 times in the HC group.LPS also increased cytokines production in both groups,while PBMCs from RA patients produced more IL-6 and TNF-α than the cells from healthy subjects (P＜0.01).Compared to the HC group,the IL-17 mRNA expression and IL-17 secretion of CD4+ CBMCs induced by the supematants of RA patients' PBMCs stimulated with LPS was significantly higher(P＜0.01 ); but there was no significant difference between the RA group and the HC group without LPS stimulation (P＞0.05).Conclusion The expression of TLR4 on PBMCs from patients with RA and its response to LPS stimulation are increased,and it has demonstrated high capability in inducing the differentiation of Th 17 cells.