在单个细胞水平上分析抗原特异性Th1/Th2细胞因子产生的关联性

Analysis of relevance of antigen-specific Th1/Th2 cytokines at a single cell level

目的 在单个细胞水平上 ,观察抗原特异性Th1和Th2细胞因子产生的关联性 ,为进一步阐明CD4 + T细胞的分化 ,细胞因子产生的相互关系及其特征提供理论依据。方法 从OVA TCR转基因小鼠的脾和淋巴结中分离CD4 + T细胞 ,在体外在抗原提呈细胞存在下 ,用卵白蛋白 (OVA)抗原多肽刺激 3d后 ,再以同样的培养条件刺激 5～ 6h ,固定细胞 ,然后进行细胞表面和细胞内细胞因子染色 ,最后利用流式细胞仪在单个细胞水平上分析Th1和Th2细胞因子产生的关联性。结果 抗原特异性CD4 + T细胞经抗原再一次刺激后 ,分泌Th1(IFN γ和IL 2 )和Th2 (IL 4、IL 5和IL 10 )细胞因子。IL 12促进IFN γ的表达 ,控制Th2细胞的分化。此外 ,大多数抗原特异性CD4 + T细胞只产生 1种细胞因子 ,1个细胞同时产生 2种细胞因子极少见。结论 在单个细胞水平上的研究结果表明 ,经抗原短暂刺激后 (3d) ,不同的CD4 + T细胞亚群只产生 1种Th1和 或Th2细胞因子 。

Objective To clarify the differentiation, cytokine production, and characterization of Th1 and Th2 cells by analyzing relevance of antigen-specific Th1 and/or Th2 cytokines at the single cell level. Methods CD4 + T cells were isolated from spleens and lymph nodes of OVA-TCR-transgenic mice and cultured with OVA peptides in the presence of antigen presenting cells. After incubation for 3 day, the cells were harvested, washed, and restimulated in the same culture conditions as described above. After restimulation for 5-6 h, the cells were fixed and stained for cell surface markers and intracellular cytokines. The cells were then measured and analyzed by fluorescence-activated cell sorting (FACS). Results After restimulation with antigen, some of the CD4 +T cells were positive either for Th1 or Th2 cytokines, but few cells was positive for both of them. In the presence of IL-12 during the primary culture, the percentage of IFN-γ + cells was markedly increased but Th2-producing cells were decreased significantly. Conclusion After stimulation with antigen, most of CD4 +T cells produce just one of cytokines and double cytokine-positive cells are rare whatever in the polarized Th1-culture condition.