成人外周血细胞中Th2功能亚群分化的体外实验研究

Studies on differentiation of Th2 functional subset among adult peripheral blood cells in vitro

目的 :研究在中性条件和Th2极化条件下CD3单克隆抗体 (CD3mAb)激活的人外周血αβT细胞Th1/Th2型细胞因子的表达。方法 :用CD3mAb、IL 2刺激人外周血单个核细胞 (PBMC) ,作为CD3mAb激活的T淋巴细胞 (CD3AT)的中性条件培养 ;用CD3mAb、IL 2、rhIL 4和抗人IL 12抗体 ,作为Th2极化条件。分别收集PBMC和培养后第 7、14和 2 1天细胞 ,加PMA、ionomycin和monensin作用 6h后 ,用多色荧光抗体标记 ,在流式细胞仪上检测αβT细胞中胞内IFN γ或IL 4产生细胞的比例。结果 :PBMC中IFN γ+ 或IL 4 + αβT细胞分别为 2 3.6 %、2 .4 %。与中性条件相比 ,Th2极化条件下培养 3周 ,αβT细胞中IFN γ产生细胞明显减少 (P <0 .0 0 1) ,IL 4 + 产生细胞显著增加 (P <0 .0 0 1) ;同时产生IFN γ+ IL 4 + 细胞从 3.6 %增加至 6 .1%。结论

Objective:To study expression of Th1/Th2 type cytokines in human peripheral αβT cells activated with CD3 monoclonal antibody(CD3mAb),cultured in neutral and Th2 type polarizing condition.Methods:Peripheral blood mononuclear cells(PBMC) were stimulated with CD3mAb to generate CD3mAb activated T cells(CD3AT);CD3AT cells were cultured in medium with IL-2 as neutral culturing condition,or with IL-4 plus anti-IL-12 neutralizing Ab as Th2 type polarizing condition for three weeks.Fresh isolation PBMC and cultured CD3AT for 7 to 21 days in neutral and Th2 type polarizing condition were stimulated with Phorbo 12-myristate 13-acetate(PMA),ionomycin and monensin.Intracellular cytokines of cell were stained with multicolor fluorescence mAb.The percentage of Th1 and Th2 expressing cell among fresh PBMC or CD3AT were measured by flow cytometry.Results:The percentages of IFN-γ and IL-4 producing T cells among fresh isolation PBMC were 23.6% and 2.4%,respectively.Compared to the neutral condition,in the culture of Th2 polarizing condition for 3 weeks,the percentages of IFN-γ + producing cells among CD3AT sharply decreased(P<0.001) and that of IL-4 producing T cells had significant increase(P<0.001),and that of IFN-γ +IL-4 +αβT cells modestly increased(3.6% to 6.1%).Conclusions:αβT cells in human peripheral blood could be developed as type-2 functional subset in Th2 polarizing condition in vitro.