体外扩增小鼠CD8^+NK1.1^+NKT细胞的研究

Mice CD8^+NK1.1^+NKT cells amplified in vitro

目的研究体外扩增小鼠CD8+NK1.1+NKT细胞的表面分子标志、分化途径及细胞属性。方法获取超抗原SEB活化后体外扩增的小鼠效应细胞,用抗CD3-PerCP、CD4-FITC、CD8-PE、NK1.1-APC、TcRVβ8-FITC和CD69-FITC荧光抗体染色后,用流式细胞仪(FCMCalibueBD,USA)鉴定CD8+NK1.1+NKT细胞的表面分子标志和分化途径。用逆转录聚合酶链反应测定细胞内各种细胞因子和Foxp3基因转录水平。结果体外扩增的细胞中91.92%是CD8+T细胞,其中22.75%的细胞是CD8+NN1.1+NKT细胞,高于正常值(0.21±0.19,n=12)108倍以上。19.61%NKT细胞是TcRVβ8+NN1.1+NKT细胞。CD69分子的表达由原始0.11%增加到85.95%。CD4+T、CD3+T细胞亚群和CD4+NKT、CD3+NKT及CD4-CD8-NKT细胞亚群没有增加。扩增细胞TGF-β的mRNA表达呈阳性,不表达Foxp3和细胞因子IL-2、4、5、6、10及IFN-γ。CD8+NKT细胞直接由CD8+T细胞分化而来。结论 CD8+NK1.1+NKT细胞的分子特征为CD69+Foxp3-TcRVβ8+TGF-β+CD8+NK1.1+;它们既不是CD8+T调节细胞(Treg.),也不是CD4+NKT细胞,直接由CD8+T细胞分化而来,带有TCRVβ8受体,应属于T细胞亚群。

Objective To study the surface markers, differentiation pathways and other properties of mice CD8^＋NK1.1^＋NKT cells amplified in vitro. Methods In vitro amplified mice CD8^＋NK1.1^＋NKT cells were obtained by treating mice spleen cells with Staphylococcus enterotoxin B （SEB）. After the cells were stained with fluorescent antibodies, their surface markers and differential pathways were detected with a flow cytometer（Calibue BD, USA）. Gene transcription levels in cytokines and Foxp3 of the cells were measured by RT-PCR. Results Of the in vitro –amplified cells, CD8^＋T cells accounted for 91.92% with 22.75% being CD8^＋NK1.1^＋ NKT cells, which was 108 times higher than that in normal lymphocytes, and NKT cells accounted for 19.61% of TcRVβ8＋ NN1.1＋NKT cells. The expression rate of CD69 was increased from 0.11% to 85.95%. The expression of CD4^＋T, CD8^＋T, CD3＋NKT, CD4^＋NKT and CD4-CD8-NKT cell subsets did not increase. The mRNA was positively expressed in amplified TGF-βcells which did not express Foxp3 and cytokines including IL-2, 4-6, 10 and IFN-γ. CD8^＋NKT cells were directly differentiated from CD8^＋T cells. Conclusion CD8^＋NK1.1^＋NKT cells, characterized by CD69＋Foxp3-TcRVβ8＋ TGF-β＋CD8^＋NK1.1^＋, are neither CD8^＋ regulatory cells（Treg,） nor CD4^＋NKT cells. They are directly differentiated from CD8^＋T cells and carry TCRVβ8 receptors, thus belonging to a subset of T lymphocytes.