γδT细胞的扩增及其信号转导分子ζ链相关蛋白-70的免疫印迹检测  

Amplification of γδT cells and detection of ZAP-70 protein via Western blotting

作　　者：王克强[1] 侯彦强[2] 朱蓓[1] 李雁[3] 陈岱韻[1] 韩国新[1] 

机构地区：[1]泰山医学院附属医院检验科, 山东泰安271000 [2]上海交通大学附属第一人民医院松江分院检验科中心实验室 [3]武汉大学医学院肿瘤研究所中南医院肿瘤科

出　　处：《中华生物医学工程杂志》2013年第6期461-463,共3页Chinese Journal of Biomedical Engineering

基　　金：山东省泰安市科技发展计划（20074043）

摘　　要：目的 检测人外周血γδT细胞的信号转导分子ζ链相关蛋白-70 （ZAP-70）的表达.方法 应用淋巴细胞分离液常规分离获取健康人外周血单个核细胞（PBMC）,用结核杆菌低分子多肽抗原（Mtb-Ag）刺激PBMC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；采用免疫印迹方法检测γδT细胞内的ZAP-70分子.结果 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％.免疫印迹显示检测到γδT细胞内相对分子质量为70 000的ZAP-70分子的表达.结论 ZAP-70分子在活化增殖的人外周血γδT细胞内高表达.Objective To detect the expression of signal transduction molecule ζ chain-associated protein 70 （ZAP-70） in proliferated γδT cells derived from human peripheral blood.Methods The peripheral blood mononuclear cells （PBMCs） were conventional separated by using lymphocyte separation liquid,and obtained from healthy volunteers.PBMCs were stimulated by low-molecular peptide antigen of mycobacterium tuberculosis （Mtb-Ag）.Following incubation for 10 days,the T cells were harvested for purification of γδT cells by using the immunomagnetic beads selection algorithm.The expression of ZAP-70 in γδT cells was detected by Western blotting.Results The proportion of γδT cells were 4.9% in fresh PBMCs,69.2% at 10 days following incubation with Mtb-Ag,and 99.3% after immunomagnetic beads selection.ZAP-70 （relative molecular mass of 70 000） expression in γδT cells was detected by Western blotting.Conclusion ZAP-70 is highly expressed in γδT cells following proliferation and activation.

关 键 词：ΓΔT细胞 ZAP-70蛋白酪氨酸激酶 结核杆菌低分子多肽抗原 印迹法 蛋白质 

分 类 号：R446.6[医药卫生—诊断学]