A20对脂多糖诱导人树突状细胞成熟度及功能的影响

Effect of A20 on the Maturity and Function of Human Dendritic Cells Induced by Lipopolysaccharide

目的探讨A20对脂多糖诱导的树突状细胞成熟度及功能的影响。方法在脂多糖诱导树突状细胞成熟过程中,沉默A20,采用定量RT-PCR检测树突状细胞中A20的表达水平,流式细胞术检测树突状细胞表面共刺激分子CD80、CD86及HLA-DR的表达水平,ELISA检测树突状细胞分泌的细胞因子IL-1β、IL-6和IL-27的水平。结果荧光显微镜下观察到绝大部分DCs被携带有GFP荧光基团标记的腺病毒转染,呈绿色荧光表达;定量RT-PCR检测显示,转染A20-shRNA腺病毒的树突状细胞A20的表达低于未转染腺病毒的树突状细胞(P<0.05);流式细胞术检测显示,沉默A20后,树突状细胞细胞表面的成熟标记分子CD80、CD86和HLA-DR的表达水平没有变化;ELISA检测显示,沉默A20的树突状细胞分泌IL-1β和IL-6的水平增加(P<0.05),而IL-27的水平降低(P<0.05)。结论携带A20-shRNA的腺病毒能够顺利转染树突状细胞,且A20对树突状细胞的成熟度无调控作用,但沉默A20后能够促进树突状细胞分泌促炎细胞因子IL-1β和IL-6,降低抑炎细胞因子IL-27的表达水平。

Objective To investigate the effect of A20 on the maturity and function of human dendritic cells induced by lipopolysaccharide.Methods During the maturation of dendritic cells induced by lipopolysaccharide,A20 was silenced.The expression level of A20 in dendritic cells was detected by quantitative RT-PCR.The expression levels of costimulatory molecules CD80,CD86 and HLA-DR on dendritic cells were detected by flow cytometry.The levels of cytokines IL-1β,IL-6 and IL-27 secreted by dendritic cells were detected by ELISA.Results Under the fluorescence microscope,most of the DCs were transfected with adenovirus carrying GFP fluorescent group,showing green fluorescence expression.Quantitative RT-PCR showed that the expression of A20 in dendritic cells transfected with A20-shRNA adenovirus was lower than that in dendritic cells not transfected with adenovirus(P<0.05).Flow cytometry showed that the expression levels of mature marker molecules CD80,CD86 and HLA-DR on the surface of dendritic cells did not change after silencing A20.ELISA showed that the levels of IL-1βand IL-6 secreted by A20 dendritic cells increased(P<0.05),while the level of IL-27 decreased(P<0.05).Conclusion Adenovirus carrying A20-shRNA can successfully transfect dendritic cells,and A20 has no regulatory effect on the maturity of dendritic cells.However,silencing A20 can promote the secretion of pro-inflammatory cytokines IL-1βand IL-6 by dendritic cells and reduce the expression of anti-inflammatory cytokine IL-27.