哮喘患儿外周血CD3^+TCRvα24^+NKT细胞亚群分析

Subpopulations of CD3^+TCRvα24^+NKT cell in PBMCs of asthma children

目的比较支气管哮喘急性发作期患儿和正常小儿外周血CD3+TCRvα24+NKT细胞频率;CD4+、CD8+、CD4-CD8-(DN)3个亚群比例;各亚群细胞胞内IL-4、IFN-γ水平及其表面活化分子CD69的表达情况。探讨NKT细胞在哮喘发作过程中的作用和机制。方法收集12例哮喘急性发作期患儿和10例健康小儿外周血,分离其中单个核细胞(PBMCs),对其表面分子CD3、TCRvα24、CD4、CD8、CD69及胞内分子IL-4、IFN-γ进行染色,流式细胞仪分析检测。结果哮喘患儿和健康小儿外周血CD3+TCRvα24+NKT细胞频率分别为0.42%、0.32%。哮喘组CD4+、CD8+、DN 3个亚群比例分别为:71.60%、14.90%、12.55%,正常对照组为:63.00%、13.12%、22.78%。哮喘患儿较正常小儿外周血CD4+NKT细胞比例上升,而DN NKT比例下降。3个亚群的NKT细胞均检测到了CD69、IL-4和IFN-γ的表达。总体而言,CD4+亚群和DN亚群胞内IL-4、IFN-γ水平较CD8+亚群高,DN NKT表面CD69表达高于其它2个亚群。但各亚群CD3+TCRvα24+NKT细胞CD69、IL-4和IFN-γ水平在哮喘组及正常对照组中未检测出明显差异。结论哮喘患儿急性发作期外周血CD3+TCRvα24+NKT细胞CD4+亚群频率上升,DN亚群频率下降。NKT细胞亚群比例的改变可能参与或介导了哮喘患儿急性期气道炎症反应。

This study designed to explore the function and mechanism of NKT cells in asthma, by comparing asthmatic and healthy children in term of the frequency of CD3＋TCRvα24＋NKT cells and the percentages of CD4＋, CD8＋ and CD4-CD8- （DN） subpopulations, as well as the expression of IL-4, IFN-γ and CD69 of every NKT subpopulation in PBMCs. Peripheral blood samples from 12 cases of acute exacerbation of asthma children and 10 cases of healthy control children were collected for preparing PBMCs, then the cell surface markers （CD3＋TCRvα24 CD4, CD8 and CD69） and intracellular cytokines （IL-4 and IFN-γ） of PBMCs were stained for flow cytometry analysis. The average percentages of CD3＋TCRvα24＋ NKT cells from blood of asthmatic and healthy children were 0.42% and 0.32% respectively. Cells could be identified based upon the expressions of CD4 and CD8 molecules. The percentage of CD4＋ NKT was 71.60%, CD8＋ NKT was 14.90% and DN NKT was 12.55% in asthmatic children compared with 63.00%, 13.12% and 22.78% in control. The results showed that children undergoing exacerbations of asthma demonstrated more CD4＋ NKT cells and less DN NKT cells as compared with healthy control children. Furthermore, we found that every subpopulation of CD3＋ TCRvct24＋ NKT could expressed CD69, IL-4, and IFN-γ. The levels of IL-4 and IFN-γ in CD4＋ and DN NKT were higher than those of CD8＋ NKT, and the expression of CD69 detected on DN NKT was more than that on the other two subpopulations. However, no significant difference was found in the expression of IL-4, IFN-γ and CD69 between asthmatic and healthy children. In conclusion, the frequency of CD3＋TCRvα24＋CD4＋ NKT cell is increased, but CD3＋TCRvα24＋DT NKT cells are decreased in blood of asthmatic children compared with healthy children, and the variation of CD3＋TCRvα24＋NKT NKT cells might involve in asthmatic attack and exacerbations.