MAPK信号转导途径在Mtb-Ag激活的γδT细胞杀伤肿瘤细胞中的作用

目的：探讨MAPK信号转导途径在结核杆菌抗原(Mtb-Ag)活化的γδT细胞杀伤肿瘤细胞中的作用。方法：用Mtb-Ag刺激正常人PBMC以诱导γδT细胞扩增，并用磁珠阳性分选法分离高纯度的γδT细胞；用MTT比色法测定γδT细胞对肿瘤细胞系K562和Raji的细胞毒活性，并观察MEK／Erk特异性抑制剂PD98059和p38 MAPK特异性抑制剂SB203580，对细胞毒活性的阻断作用：用流式细胞仪检测肿瘤细胞诱导γδT细胞上CD69的表达，并观察PD98059和SB203580对CD69表达的抑制作用。结果：新鲜分离的PB-MC，用Mtb-Ag刺激培养第10天，用磁珠阳性法分离的细胞中γδT细胞的比率，分别为3．56％、74．63％和98．20％。PD98059可抑制γδT细胞的杀瘤活性，且对γδT细胞杀伤Fas低表达的K562细胞的抑制率(39．27％)高于对γδT细胞杀伤高表达Fas的Raji细胞的抑制率(26．58％)。PD98059还能明显抑制肿瘤细胞诱导γδT细胞表达CD69；而SB203580对γδT细胞的杀瘤活性和肿瘤细胞诱导的CD69的表达均无影响。结论：MAPK途径中的Erk通路(而不是p38通路)参与了γδT细胞对肿瘤细胞细胞毒活性的启动，且可能对γδT细胞的颗粒外吐作用较大，而对Fas／FasL介导的细胞毒活性的作用较小。MAPK途径的Erk通路可能还参与肿瘤细胞诱导γδT细胞的活化。

The role of MAPK signal transduction pathway in killing tumor cells by Mtb-Ag activated γδT cells

AIM: To explore the role of MAPK signal transduction pathway in killing tumor cells by Mycobacteria tuberculosis antigen (Mtb-Ag) activated human gammadeltaT cells. METHODS: Normal human peripheral blood mononuclear cells (PBMCs) were stimulated with Mtb-Ag and expanded in rIL-2-containing medium to induce gammadeltaT cells. The highly purified gammadeltaT cells were isolated by positive selection with MACS separator. Proportion of gammadeltaT cells was detected by flow cytometry (FCM). The cytotoxicity and CD69 expression of gammadeltaT cells pre-treated with or without PD98059 (Erk inhibitor) or SB203580 (p38 inhibitor) were detected by MTT colorimetry assay and FCM, respectively. RESULTS: The percentages of gammadeltaT cells in freshly isolated PBMCs, Mtb-Ag-activated PBMCs and cells sorted by MACS were 3.56%, 74.63% and 98.20%, respectively. The cytotoxicities of gammadeltaT cells to tumor cell lines (K562 and Raji cells) were inhibited by PD98059. The inhibitory rate of cytotoxicity to K562 cells by gammadeltaT cells (39.27%) was higher than that to Raji cells(26.58%). CD69 expressions on gammadeltaT cells induced by K562 or Raji cells were decreased by pretreatment with 100 mumol/L of PD98059. But SB203580 had no effects on the cytotoxicity and CD69 expression on gammadeltaT cells induced by K562 and Raji cells. CONCLUSION: Erk MAPK pathway, not p38 MAPK, plays an important role in triggering cytotoxicity of gammadeltaT cells. The Erk pathway may affect on the granule exocytosis of gammadeltaT cells more than the Fas/FasL-mediated cytotoxicity of gammadeltaT cells. Moreover, Erk MAPK pathway is also involved in expression of activation molecule CD69 on gammadeltaT cells induced by tumor cells.