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目的比较卡介苗(bacillus Calmette-Guerin vaccine,BCG)与结核分枝杆菌早期分泌靶抗原(early secreted antigenic target-6,ESAT-6)刺激人外周血γδT细胞分泌细胞因子的能力。方法采用Ficoll密度梯度离心法分离健康人外周血单核细胞(peripheral blood mononuclear cell,PBMC),加入Anti-human gamma-delta TCR-FITC抗体标记γδT细胞,上流式细胞仪进行分离纯化;分别用BCG和ESAT-6刺激γδT细胞,同时,以不加任何刺激因子的γδT细胞作为空白对照,分别于刺激培养后第1、3、6、9和12天取上清,采用ELISA试剂盒检测IL-17、TNF-α及IFNγ的分泌水平;上流式细胞仪检测γδT细胞的增殖水平。结果γδT细胞占PBMC的4.83%,其纯度为88.90%;与空白对照组比较,BCG和ESAT-6刺激的γδT细胞分泌的IL-17、TNF-α及IFNγ水平均明显升高,且ESAT-6组明显高于BCG组(P均<0.05);与空白对照组相比,BCG组和ESAT-6组γδT细胞数量明显增加,且ESAT-6组较BCG组增加明显(P均<0.05)。结论 BCG和ESAT-6均可刺激γδT细胞增殖,并大量分泌IL-17、TNF-α及IFNγ,且ESTA-6的刺激作用强于BCG。
Objective To compare the ability of cytokines secreted by human peripheral blood γδT cells stimulated by bacillus Calmette-Guerin vaccine (BCG) and early secreted antigenic target-6 (ESAT-6). Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers by Ficoll density gradient centrifugation. Anti-human gamma-delta TCR-FITC antibody labeled γδT cells were isolated and purified by flow cytometry. And ESAT-6 stimulated γδT cells. At the same time, γδT cells without any stimulation factors were used as blank control, and supernatants were harvested on days 1, 3, 6, 9 and 12 after stimulated culture. ELISA kit was used to detect IL- 17, the secretion of TNF-α and IFNγ; the proliferation of γδT cells was detected by flow cytometry. Results γδT cells accounted for 4.83% of PBMCs with a purity of 88.90%. Compared with the blank control group, the levels of IL-17, TNF-α and IFNγ secreted by BCG and ESAT-6 stimulated γδT cells were significantly increased, and ESAT- (P <0.05). Compared with the blank control group, the number of γδT cells in BCG group and ESAT-6 group increased significantly, and the ESAT-6 group increased significantly compared with BCG group (all P <0.05) . Conclusion Both BCG and ESAT-6 can stimulate the proliferation of γδT cells and secrete more IL-17, TNF-α and IFNγ, and the stimulation of ESTA-6 is stronger than that of BCG.