CpG ODN对Treg细胞及Th17细胞分化的影响

Effect of CpG ODN on the Differentiation of Treg/Th17 Cells

目的研究免疫佐剂CpG ODN对小鼠脾细胞中Treg细胞、Th17细胞分化的影响,为进一步的临床免疫治疗应用奠定基础。方法体外分离小鼠脾细胞后,加入抗CD3单抗及抗CD28单抗培养,并相应加入诱导Treg细胞分化的细胞因子TGF-β、IL-2或诱导Th17细胞分化的细胞因子TGF-β、IL-6、IL-23,培养24h后加入CpG1982、CpG1826继续培养48h,培养结束后收集细胞进行细胞内染色流式细胞术检测Treg/Th17细胞,利用实时定量PCR检测Foxp3、RORγt、IL-10、IL-17mRNA的水平,Western blot检测Foxp3、RORγt蛋白表达,ELISA检测脾细胞培养上清IL-10、IL-17水平。结果在细胞因子TGF-β、IL-2的诱导下,CD4+Foxp3+Treg细胞数量明显增多,核转录因子Foxp3 mRNA和蛋白水平均增高,且细胞培养上清中IL-10的水平也升高,在细胞因子诱导的同时加入CpG1826培养后CD4+Foxp3+Treg细胞数量明显减少,Foxp3水平降低,培养上清中IL-10的水平降低,差异具有统计学意义(均P<0.05);单独加入CpG1826以及在培养液中加入细胞因子TGF-β、IL-6、IL-23后,Th17细胞数量均明显增加,核转录因子RORγt水平升高,细胞培养上清中IL-17水平增加,与对照组相比差异均有统计学意义(均P<0.05),CpG1826联合细胞因子组则对Th17细胞分化具有更加明显的刺激效应(P<0.05)。结论免疫佐剂CpG1826具有抑制小鼠脾细胞中Treg细胞分化、促进Th17细胞分化的作用,从而发挥对Treg/Th17系统的调节功能。

Objective To examine the effects of CpG ODN on differentiation of Treg/Th17 cells in vitro.Methods Mouse spleen cells separated in vitro were cultured with anti-CD3 and anti-CD28 monoclonal antibody and TGF-β,IL-2or TGF-β,IL-6,IL-23.After 24 h,CpG1982or CpG1826 was added to the culture system.Treg/Th17 cells were detected by flow cytometry（FCM）after collecting spleen cells.The mRNA expression levels of Foxp3,RORγt,IL-10 and IL-17 were measured by real time PCR and the protein expression levels of Foxp3 and RORγt were measured by Western blotting.The levels of IL-10 and IL-17 in supernatant were measured by ELISA.Results The number of CD4＋Foxp3＋Treg cells increased significantly in the presence of TGF-βand IL-2（P〈0.05）.The mRNA and protein levels of Foxp3 and IL-10 in the cell culture supernatant were significantly increased（P〈0.05）.CD4＋Foxp3＋Treg cells were reduced significantly after adding CpG1826（P〈0.05）.The levels of Foxp3 and IL-10 significantly decreased（P〈0.05）.The number of Th17 cells was increased significantly in the presence of CpG1826,or TGF-β,IL-6and IL-23（P〈0.05）.The levels of RORγt and IL-10 in the cell culture supernatant were increased（P〈0.05）.There was a more significant stimulating effect of CpG1826 combined with TGF-β,IL-6and IL-23 on Th17cells（P〈0.05）.Conclusion CpG1826 can regulate Treg/Th17 cells in vitro through inhibiting differentiation of Treg cells and stimulating differentiation of Th17cells