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

    • 摘要: 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21天细胞,加PMA、ionomycin和monensin作用6 h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23.6%、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<0.001),IL-4+产生细胞显著增加(P<0.001);同时产生IFN-γ+IL-4+细胞从3.6%增加至6.1%。结论: 体外Th2极化条件可以使人外周血αβT细胞分化为Th2功能亚群。

       

      Abstract: 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.

       

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