摘要目的探讨丝裂原活化蛋白激酶/细胞外信号调节激酶（MAPK/ERK）特异性抑制剂PD98059对结核杆菌低分子多肽抗原（Mtb?Ag）活化γδΤ细胞的影响。方法分离获取健康人外周血单个核细胞（PBMC），分别使用佛波醇酯+离子霉素（PMA+IM）、Mtb?Ag刺激PBMC，流式细胞术检测处理0、6、12、24、48和72 h后γδΤ细胞CD69的表达。浓度为0、1、10和100μmol/L的PD98059预处理PBMC后再分别加入Mtb?Ag（Mtb?Ag组）、PMA+IM（PMA+IM组）处理24 h，流式细胞术检测活化γδΤ细胞CD69的表达；Mtb?Ag组继续培养10 d后收集细胞计数细胞总数、检测γδΤ细胞比例并计算γδΤ细胞的绝对数量。结果使用PMA+IM处理6 h，γδΤ细胞CD69表达达高峰；使用Mtb?Ag处理24 h，γδΤ细胞CD69表达达高峰。处理后同一时点两组γδΤ细胞CD69表达比较，差异均具有统计学意义（均P<0.05）。使用终浓度分别为0、1、10和100μmol/L的PD98059预处理PBMC后再分别加入Mtb?Ag、PMA+IM处理24 h，流式细胞术检测显示Mtb?Ag组γδΤ细胞CD69表达分别为（79.0±0.8）%、（75.0±0.7）%、（54.0±0.5）%和（17.0±0.2）%，而PMA+IM组γδΤ细胞CD69表达均在98%以上；Mtb?Ag组继续培养10 d后，细胞总数由培养前的1.5×106分别增殖到（10.3±2.5）×106、（9.5±2.1）×106、（5.8±1.8）×106和（2.1±0.5）×106，γδΤ细胞数分别增殖到（6.2±0.9）×106、（5.0±0.8）×106、（2.1±0.5）×106和（0.4±0.1）×106。结论 Mtb?Ag可以通过MAPK/ERK信号转导通路特异性活化γδΤ细胞。

abstractsObjective To investigate the effect of mitogen activated protein kinase/extracellular signal?regulated kinase(MAPK/ERK)specific inhibitor PD98059 on low molecular polypeptide antigen of Mycobacterium tuberculosis (Mtb?Ag) activated γδΤcells. Methods Peripheral blood mononuclear cells (PBMCs)were isolated from healthy volunteers,and stimulated with phorbol myristate acetate+ionomycin (PMA+IM)and Mtb?Ag,respectively. Flow cytometry was used to determine the expressions of CD69 inγδT cells at 0,6,12,24,48 and 72 h. After pretreatment with PD98059 at the concentrations of 0,1,10 and 100μmol/L,PBMCs were treated with Mtb?Ag(Mtb?Ag group)and PMA+IM(PMA+IM group)for 24 h, respectively. Flow cytometry was used to determine the expression of CD69 in the activatedγδT cells. At 10 d after the culture,the total number of cells was collected,the proportion of γδT cells was examined,and absolute number of γδT cells was calculated in the Mtb?Ag group. Results At 6 h after the treatment with&nbsp;PMA+IM,the expression of CD69 inγδΤcells reached the peak. At 24 h after the treatment with Mtb?Ag, the expression of CD69 inγδΤcells reached the peak. At the same time point after the treatment,there were statistically significant differences in the CD69 expression ofγδT cells between the two groups(all P<0.05). Pretreated with PD98059 at the final concentrations of 0,1,10 and 100μmol/L,PBMCs were treated with Mtb?Ag and PMA+IM for 24 h,respectively. The expression of CD69 inγδT cells in the Mtb?Ag group was (79.0±0.8)%,(75.0±0.7)%,(54.0±0.5)%and(17.0±0.2)%,respectively,whereas those in the PMA+IM group were all more than 98%;At 10 d after the culture in the Mtb?Ag group,the total number of cells reached(10.3±2.5)×106,(9.5±2.1)×106,(5.8±1.8)×106 and(2.1±0.5)×106 from 1.5×106,and the number of γδΤ cells reached(6.2 ± 0.9)× 106,(5.0 ± 0.8)× 106,(2.1 ± 0.5)× 106 and(0.4 ± 0.1)× 106,respectively. Conclusion Mtb?Ag may specifically activateγδT cells through MAPK/ERK signal transduction pathway.