T细胞的扩增及其信号转导分子ZAP-70的检测

The detection of signal transduction molecular ZAP-70 of γδT cells

目的检测Y6T细胞信号转导分子ξ链相关蛋白-70（ξ-chain associated protein70，ZAP-70）。方法分离获取健康人PBMC，用结核杆菌低分子多肽抗原（Mtb-Ag）刺激PBMC，通过流式细胞仪检测总T细胞和Y6T细胞CD69分子的动态表达；Mtb-Ag活化γδT细胞增殖培养，10d后收集细胞，用免疫磁珠阳性分选法分离获取高纯度的γδT细胞；western blot方法检测俩T细胞内的ZAP-70分子。结果总T细胞和γδT细胞均在活化刺激24h时表达CD69分子达高峰，但总T细胞仅为16％，γδT细胞可达75．2％；新鲜分离的PBMC中γδT细胞的比例仅为4．9％，Mtb-Ag刺激培养10d后升为69．2％，免疫磁珠阳性分选后达99．3％；检测γδT细胞内的ZAP-70分子。结论Mtb—Ag可特异性激活γδT细胞，用Mtb-Ag刺激γδT细胞活化增殖培养，可获得大量的γδT细胞；成功地检测到细胞内ZAP-70分子，这为γδT细胞内其它分子的检测分析奠定方法学基础，也为进一步检测γδT细胞活化信号转导过程中ZAP-70分子的激活及作用奠定基础。

Objective To detect signal transduction molecular ξ-ehain associated protein 70 （ZAP-70） of γδT cells. Methods Peripheral blood mononuclear lymphocytes （PBMCs） were isolated from peripheral blood of healthy subjects and stimulated in vitro with Mycobacterium tuberculosis low molecular peptide antigen （Mtb-Ag）, CD69 expression of total T cells and γδT cells were measured by flow cytometry at different hours after stimulation, and the number of expanded γδT cells were counted after 10 days of culture, γδT cells coated by TCR78 Hapten-antibody in combination with MACS anti-Hapten microbeads-FITC were separated with a LS＋ positive selection macs separator, the proportion of 78 T cells were measured by anti-TCRγδ-PE staining and flow cytometry, the ZAP-70 molecular of γδT cells were detected by western blot. Results The proportions of CD69 expressing cells in total T cells and γδT ceils after 24h stimulation of PBMC with Mtb-Ag were 16% and 75.2%, respectively; the proportion of γδT cells in fresh PBMC is 4.9%, then increased to 69.2% after 10 days of Mtb-Ag stimulation and to 99.3% after magnetic purification; the ZAP-70 molecular in γδT cells could be detected by western blot. Conclusions Mtb-Ag is a specific stimulator for activation of γδT cells, it is effective technique to generate a large number of γδT cells from PBMC that stimulated by Mtb-Ag and expanded in IL-2 containing medium in vitro, the ZAP-70 molecular of γδT cells could be successfully detected, this approach provided a technique support for further study in the signal transduction of ZAP-70 of γδT cells.