鞭毛蛋白与IL-12协同诱导人NK细胞产生IFN-γ机制的探讨

The synergistic effect of flagellin and interleukine-12 on the induction of IFN-γ production from human NK cells and its mechanism

目的:探讨细菌鞭毛蛋白(flagellin)与IL-12协同诱导人NK细胞产生IFN-γ的作用和机制。方法:自正常人静脉血中分离PBMCs,分别与培养液(medium)、flagellin、IL-12或flagellin+IL-12共同培养。三天后,利用酶联免疫吸附法(ELISA法)检测培养上清中IFN-γ的水平。收集初次培养组细胞,洗去刺激后进行二次培养,检测IFN-γ的产生。利用流式细胞仪检测NK细胞上IL-12Rβ1和TLR5的表达情况。结果:一次培养的结果表明,flagellin或IL-12单独刺激PBMCs可诱导较低水平IFN-γ的产生。共同刺激下,可协同诱导高水平IFN-γ产生。二次培养结果表明,不同剂量IL-12的刺激flagellin初次培养组细胞,IFN-γ的产生呈剂量依赖性的增加;IL-12初次刺激组细胞在flagellin的二次刺激下,IFN-γ的产生亦呈剂量依赖性增加。流式结果表明,flagellin可上调NK细胞IL-12Rβ1的表达。IL-12可上调NK细胞TLR5的表达。结论:flagellin通过上调NK细胞IL-12Rβ1的表达,IL-12通过促进NK细胞TLR5的表达,从而协同诱导IFN-γ的产生。

Objective：To study the synergic effect of flagellin and interleukine-12（IL-12） on the production of IFN-γ by human NK cells and its mechanism.Methods：PBMCs isolated from normal donors were cultured with medium,flagellin,IL-12 or flagellin plus IL-12.Culture supernatants were collected at day 3 and the level of IFN-γ was measured by ELISA.Cells under primary culture conditions were collected,washed,restimulated,and the production of IFN-γ was detected after 3 days.The expression of IL-12 receptor β1 and toll-like receptor 5 on NK cell subsets was detected and analyzed by flow cytometry.Results：In the primary culture conditions,application of suboptimal dose of flagellin or IL-12 could induce low levels of IFN-γ.Stimulation of PBMCs with flagellin plus IL-12 could synergistically induce IFN-γ production.Under the secondary culture conditions,cells that pre-treated with flagellin were restimulated with different concentrations of IL-12,which could induce high levels of IFN-γ in a dose-dependent manner.Cells pre-stimulated with IL-12 could,in a dose-dependent manner,enhance the production of IFN-γ in the presence of different concentrations of flagellin.Flow cytometry analysis indicated that the expression of IL-12 receptorβ1 on NK cell subsets was upregulated when stimulated with flagellin and IL-12 could enhance the expression of toll-like receptor 5 on NK cell subsets.Conclusion：Up-regulation of IL-12 receptorβ1 by flagellin and enhanced expression of the toll-like receptor 5 by IL-12 contribute to the synergistic effects of IFN-γ production by NK cell subsets.