摘要
目的 观察结核杆菌抗原 (Mtb Ag)活化的γδT细胞信号转导分子ZAP 70酪氨酸磷酸化的特点。方法 用Mtb Ag和抗CD3单克隆抗体 (CD3McAb)体外刺激人外周血单个核细胞(PBMC) ,诱导活化 ,在含IL 2的培养液中扩增培养 ,分别获得Mtb Ag激活的T细胞 (MtbAT)和CD3McAb激活的T细胞 (CD3AT)。再分别用Mtb Ag和CD3McAb刺激 ,在不同时间内提取细胞裂解液 ,用抗 酪氨酸磷酸化蛋白抗体作免疫沉淀 ,用抗ZAP 70单克隆抗体进行免疫印迹。结果 MtbAT中γδT细胞可达 6 0 %～ 80 % ,CD3AT中CD3+ 细胞可达 90 %以上 ,而γδT细胞 <10 % ;MtbAT细胞用Mtb Ag再刺激后 15min时出现ZAP 70的酪氨酸磷酸化 ,30min达高峰 ,可持续到 6 0～ 12 0min ,而CD3AT用CD3McAb再刺激后仅在 5min时检测到ZAP 70的磷酸化。结论 与CD3McAb对普通T细胞的作用比较 ,Mtb Ag刺激可优先活化γδT细胞 ,其对γδT细胞ZAP 70活化的特点是 :较慢、较强。
Objective To observe the dynamics of phosphorylation of ZAP-70 in γδT cells activated by Mycobacterium tuberculosis antigen (Mtb-Ag). Methods PBMC were stimulated with Mtb-Ag or CD3 McAb, cultured in IL-2 containing media, to generate Mtb-Ag activated T cells (Mtb AT) and CD3 McAb activated T cells (CD3 AT), respectively. The Mtb AT and CD3 AT were re-stimulated with Mtb-Ag and CD3 McAb, respectively. The cell lysates from re-stimulated T cells were precipitated by phosphotyrosine monoclonal antibody ((p-Tyr-100)) and the tyrosine phosphorylated ZAP-70 in the precipitants was detected by immunoblotting with anti-ZAP-70 monoclonal antibody. Results The percentages of γδT cells in Mtb AT and αβT cells in CD3 AT were 60%-80% and 90%, respectively. The tyrosine phosphorylation of ZAP-70 was detected in Mtb AT that were re-stimulated with Mtb-Ag at 15 min, reached climax at 30?min, and maintained for 60-120?min, while the tyrosine phosphorylation of ZAP-70 was detected in CD3 AT that were re-stimulated with CD3 McAb only at 5?min. Conclusion γδT cells could be preferentially activated by Mtb-Ag. The characteristics of activation of signal molecules in γδT cells induced by Mtb-Ag were slow, more powerful and longer duration than those of αβT cells induced by CD3 McAb. [
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2004年第11期874-877,共4页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目 (No .3 0 0 70 72 1)
安徽省自然科学基金资助项目 (No .990 44 5 42 )
