巨型艾美耳球虫Th1类细胞因子刺激性分子EmARM-β对鸡PBMC和T细胞亚群免疫功能的影响

Effects of EmARM-β,Eimeria maxima Th1 cytokine stimulatory molecule on immune function of chicken PBMC and T cell subsets

[目的]本文旨在探讨巨型艾美耳球虫Th1类细胞因子刺激性分子Armadillo/β-catenin样重复序列蛋白(EmARM-β)对鸡免疫细胞功能的影响。[方法]利用密度梯度离心法和免疫磁珠分选法分别分离出鸡外周血单个核细胞(peripheral blood mononuclear cell,PBMC)、CD8^(+)T细胞和CD4^(+)CD25^(-)T细胞,之后将EmARM-β重组蛋白(rEmARM-β)分别与上述细胞进行体外共孵育6 h,利用CCK-8试剂和qPCR法分别检测并分析其对上述细胞增殖能力及相关细胞因子分泌水平的影响。[结果]经免疫磁珠分选后,流式细胞术检测鸡CD8^(+)T细胞和CD4^(+)CD25^(-)T细胞的纯度分别为93.01%和88.88%。与对照组相比,rEmARM-β显著促进鸡PBMC的增殖能力,显著上调Th1类细胞因子(ifn-γ和il-2)和促炎性细胞因子(il-1β、il-6和il-17a)mRNA水平;显著促进CD8^(+)T细胞的增殖能力,显著上调其ifn-γ、il-2和tnf-α mRNA水平,显著上调穿孔素、fasl(Fas配体)和fas mRNA水平;能显著促进CD4^(+)CD25^(-)T细胞的增殖能力,显著上调其ifn-γ和il-2 mRNA水平,下调il-4和il-10 mRNA水平。[结论]EmARM-β可以有效促进鸡PBMC和T细胞亚群(CD8^(+)T细胞和CD4^(+)CD25^(-)T细胞)的增殖能力,提升其Th1类细胞因子和促炎性细胞因子的分泌水平,提示EmARM-β具有研发鸡球虫病新型疫苗候选抗原的潜力。

[Objectives]This study aimed to investigate the effect of Armadillo/β-catenin like repeat containing protein(EmARM-β),a Th1 cytokine stimulatory molecule from Eimeria maxima on functions of chicken immune-related cells.[Methods]In this study,chicken peripheral blood mononuclear cell(PBMC),CD8~+T cells,and CD4~+CD25~-T cells were isolated by density gradient cell centrifugation method and immunomagnetic bead sorting method,respectively.Subsequently,EmARM-β recombinant protein(rEmARM-β)was incubated with the above cells in vitro,CCK-8 solution and qPCR were used to detect and analyze the effects on the proliferation ability of the above cells and the secretion levels of cytokines,respectively.[Results]The results showed that purity of chicken CD8~+T cells and CD4~+CD25~-T cells were 93.01% and 88.88%,respectively.rEmARM-β significantly enhanced the proliferation capacity,upregulated Th1 type cytokines(ifn-γ,and il-2)and proinflammatory cytokines(il-1β,il-6,and il-17a)mRNA levels in chicken PBMC compared with the control group.Effective stimulations of rEmARM-β on the cell proliferation capacity and mRNA levels of ifn-γ,il-2,tnf-α,perforin,fasl,and fas in chicken CD8~+T cells were also observed.The molecule significantly promoted cell proliferation capacity,upregulated the mRNA levels of ifn-γ and il-2,and downregulated the mRNA levels of il-4 and il-10 in chicken CD4~+CD25~-T cells.[Conclusions]EmARM-β,the Th1 cytokine stimulatory molecule,could effectively promote cell proliferation,secretions of Th1 cytokines and proinflammatory cytokines of chicken PBMC and T cell subsets,suggesting the EmARM-β is the potential candidate antigen to develop novel vaccine against chicken coccidiosis.