纤连蛋白对人外周血单核细胞TLR4介导的脂多糖反应的影响

摘要

目的 研究纤连蛋白(Fn)刺激对人外周血单核细胞( PBMCs)炎症因子释放和Toll样受体4(TLR4)表达的影响.方法 采用梯度离心法从浓缩人外周血白细胞中分离出PBMCs,设立空白对照组、Fn刺激组、LPS刺激组以及Fn+ LPS复合刺激组,分别刺激PBMCs 0.5、2、4、8、12h后收集细胞培养上清,采用酶联免疫法(ELISA)检测其炎症因子人白介素6(IL-6)、人白介素10(IL-10)及肿瘤坏死因子α(TNF-α)的水平；采用Western blotting法检测各刺激物对TLR4表达的影响.结果 4、8h复合刺激组IL-6释放量分别为727.00±37.73和918.25±26.78pg/ml,明显高于同时间点其他3组(P＜0.05)；2h复合刺激组TNF-α释放量明显增加,且高于同时间其他3组(P＜0.05)；0.5、2、4h复合刺激组IL-10释放量呈缓慢递增,8、12h分别达784.74±91.96、1081.74±57.98pg/ml,与同时间其他3组比较明显增加(p＜0.05).Westem blotting结果显示,1,2h复合刺激组TLR4的表达水平明显高于各时间点的其他3组(P＜0.05).结论 单纯Fn或LPS刺激即可促进PMBCs TLR4表达上调,Fn复合LPS刺激可进一步增强TLR4的表达,并导致炎症介质IL-6、TNF-α释放的显著增加,提示Fn可增强PMBCs对LPS刺激的敏感性.%Objective The present study investigates the impact of fibronectin (Fn) on the inflammatory factor release of human peripheral blood mononuclear cells (PBMCs) and TLR4 expression. Methods Gradient centrifugation was used to separate normal PBMCs from concentrated human PBMCs. The blank control. Fn stimulus. LPS stimulus, and Fn plus LPS complex stimulus groups were established to stimulate the PBMCs for0. 5, 2, 4, 8. and 12 h, respectively. Subsequently, cell culture supernatant samples were collected to test the inflammatory factors IL-6, IL-10, and TNF-o using ELISAs, and Western blot was used to detect the effect of every stimulus on Toll-like receptor 4 (TLR4) expression. Results At the 4 and 8 h time points, the IL-6 production in the Fn plus LPS group reached 727+37. 73 and 918.25±26. 78 pg/ml, respectively, which were much higher than those in the other groups (P<0.05). Moreover, the TNF-a production in the Fn plus LPS group increased 2 h after stimulation and was significantly higher than those in the other three groups (P<0.05). The IL-10 productions in the Fn plus LPS group showed a slowly increasing trend at the 0. 5. 2, and 4 h time points and reached 784. 74+91. 96 and 1081. 74±57. 98 pg/ml at the 8 and 12 h time points, respectively. Again, these results were significantly higher than those in the other groups (P<0. 05). The Western blot analysis showed that the expression level of TLR4 in the complex stimulus group was evidently higher than those in the other three groups at each time point (P< 0. 05). Conclusion Fn or LPS stimulation can therefore promote the expression of TLR4 in PBMCs. However, their combination can remarkably increase the expression of TLR4 and the production of IL-6 and TNF-o. These results imply that Fn can enhance the sensitivity of PBMCs to LPS.