锌指蛋白A20抑制Toll样受体4介导的人单核细胞炎症反应的实验研究

Zinc finger protein A20 gene transfection inhibits inflammatory reaction mediated by TLR-4 in human monocytes

目的:观察锌指蛋白A20过度表达对人单核细胞膜Toll样受体(TLR4),下游促炎因子肿瘤坏死因子-α(TNF-α)与白细胞介素(IL)-12,以及抗炎因子IL-10表达的影响,探讨锌指蛋白A20对单核细胞炎症反应的抑制作用及可能的调节机制。方法:Ficoll细胞分离液分离人外周血单核细胞,随机分为对照组、脂多糖(LPS)组、A20转染组与LPS+A20转染组。荧光显微镜检测GFP报告基因,免疫组织化学检测A20蛋白的表达,RT-PCR检测A20及TLR4的mRNA表达,流式细胞检测技术检测TLR4的蛋白表达,ELISA方法检测上清液TNF-α、IL-12及IL-10表达水平。结果:LPS刺激后,单核细胞TLR4和内源性A20的mRNA和蛋白、TNF-α、IL-12和IL-10表达较对照组均明显升高(均P<0.01);TNF-α/IL-10和IL-12/IL-10均明显高于对照组(均P<0.01);转染A20基因的单核细胞,在无LPS刺激的条件下,上述指标与对照组相比均差异无统计学意义;转染A20基因的单核细胞在LPS刺激后,TLR4mRNA和蛋白、TNF-α、IL-12的表达以及TNF-α/IL-10和IL-12/IL-10均显著低于LPS组,而IL-10的表达明显高于对照组和LPS组(均P<0.01)。结论:TLR4激活介导单核细胞的炎症反应,且正反馈调节其自身受体TLR4和内源性A20的表达;A20参与单核细胞TLR4激活所介导的炎症反应,其表达增加与TLR4表达的增加有关;单纯提高A20表达对未被激活的单核细胞TLR4及其信号通路影响不大;A20过表达可抑制TLR4激活所介导的单核细胞的炎症反应。

Objective：To explore the protection and potential mechanism of zinc finger protein A20 on human monocytes inflammatory reaction. Method：Monocytes were separated by Ficoll from peripheral blood and randomly divided into 4 groups as following： ①control group,②LPS-stimulated group,③A20-transfected group and,④LPS-stimulated ＋A20-transfected group.Immunofluorescence and immuno-histochemistry were employed to analyze the expression of green fluorescent protein（GFP） and zinc finger protein A20 respecitively.RT-PCR was used to detected TLR4 and A20 mRNA expression.Flow cytometry was used to determine the expression of TLR4 in monocytes.The levels of tumor necrosis factor（TNF）-α,interleukin（IL）-12 and IL-10 in the supernatants were measured by ELISA. Result：After stimulated with LPS,the expression of TLR4,A20,TNF-α,IL-12 and IL-10 increased significantly in the monocytes（P〈0.01）.The ratio of TNF-α/IL-10 and IL-12/IL-10 also elevated（P〈0.01）.Compared to control group,transfection with exogenous A20 gene alone without LPS stuimulation didn’t influence the indexes in the monocytes that we have measured as aboved.In A20-transfected and LPS-stimulated monocytes,the expression of TLR4,TNF-α,IL-12 and the ratio of TNF-α/IL-10 and IL-12/IL-10 were lower than that in LPS-stimulated monocytes,while the expression of IL-10 was higher than that in control and LPS-stimulated monocytes. Conclusion：TLR4 mediates the inflammatory reaction of monocyte,and up-regulate the expression of TLR4 and endogenous protein A20 by positive feedback.A20 mediate the inflammatory reaction after TLR4 activation,and its expression is positively correlated with TLR4 expression.Overexpression of A20 in monocyte suppress the inflammatory reaction after TLR4 activation.