人脐带血与周围血中NKT细胞频率、亚群、表型及功能的比较

Comparison of frequencies,subpopulations,phenotypes,biological functions of human NKT cells from cord blood mononuclear cells and peripheral blood mononuclear cells

目的比较足月产新生儿脐带血和正常人周围血NKT细胞的频率、亚群、表型特征及功能。方法分离足月产新生儿脐带血CBMCs和正常成年人周围血PBMCs,流式细胞术检测TCRvβ11、CD4、CD8、CD45RA、CD62L、CCR7等表面分子的表达及细胞因子IL-4和IFN-γ的产生。结果 CBMCs和PBMCs中CD3+TCRvβ11+NKT细胞的平均频率分别为0.35%和0.33%,二者无显著差别。CBMCs中CD4+NKT细胞频率(67.39%)高于PBMCs中CD4+NKT细胞频率(54.08%),但CD8+NKT细胞,PBMCs明显高于CBMCs(22.35%对5.86%),CD4-CD8-NKT二者相差无统计学意义。CBMCs中CD3+TCRvβ11+CD45RA+NKT细胞的频率(88.37%)高于PBMCs(61.32%)。CBMCs中CD62L(56.66%对49.60%)和CCR7(22.64%对20.03%)的比例稍高于PBMCs,但其差别无显著性。未刺激CBMCs和PBMCs中CD3+TCRvβ11+NKT细胞均不产生IL-4和IFN-γ,PMA+Ionomycin刺激后,CBMCs中CD3+TCRvβ11+NKT细胞仍不能产生IL-4和IFN-γ,但PBMCs中CD3+TCRvβ11+NKT细胞却有6.74%产生IL-4、26.96%产生IFN-γ、1.26%同时分泌IL-4和IFN-γ,产生细胞因子的CD3+TCRvβ11+NKT细胞主要为记忆性NKT细胞。结论 CBMCs和PBMCs中CD3+TCRvβ11+NKT细胞的亚群、表型和功能均存在一定差别,记忆性NKT细胞可能与其再次免疫应答相关。

This study is aimed to compare the characteristics of NKT cells from neonatal cord blood mononuclear cells（CBMCs） and peripheral blood mononuclear cells（PBMCs）.We isolated CBMCs and PBMCs,and then detected cell surface markers（CD3,TCRvβ11,CD4,CD8,CD45RA,CD62L,CCR7） and then productions of IL-4 and IFN-γ after stimulation with PMA plus ionomycin.Finally,we identified the different characteristics of CD3＋TCRvβ11＋ NKT cells from CBMCs and PBMCs.There was no significant difference between the mean frequency of CD3＋TCRvβ11＋ NKT cells in CBMCs（0.35%） and in PBMCs（0.33%）.The frequency of CD4＋ NKT cells from CBMCs（67.39%） was higher than that from PBMCs（54.08%）,whereas the frequency of CD8＋ NKT cells from PBMCs（22.35%） was evidently higher than that from CBMCs（5.86%）,and there was no significant difference between the frequency of CD4-CD8-NKT cells from CBMCs and PBMCs.The frequency of CD3＋TCRvβ11＋CD45RA＋ NKT cells from CBMCs（88.37%） was higher than that from PBMCs（61.32%）.Although the trend of expressions of CD62L and CCR7 was the same as CD45RA,there was no significant difference between them.CD3＋TCRvβ11＋ NKT cells from both CBMCs and PBMCs could not produce IL-4 and IFN-γ without stimulation.After stimulation with PMA plus ionomycin,CD3＋TCRvβ11＋ NKT cells from CBMCs still could not secret IL-4 or IFN-γ,however,6.74% of CD3＋TCRvβ11＋ NKT cells from PBMCs could produce IL-4,26.96% of them produced IFN-γ and 1.26% of them produced both IL-4 and IFN-γ.Results also proved that most of cytokines were produced by memory CD3＋TCRvβ11＋ NKT cells.We concluded that there are some differences in the characteristics of CD3＋TCRvβ11＋ NKT cells from CBMCs and PBMCs,and the secondary immune responses may be related to memory NKT cells.