PKM2是TCRγδ的配体吗?

Is PKM2 a ligand of TCRγδ?

目的检验M2型丙酮酸激酶(PKM2)是否是肿瘤细胞表面TCRγδ识别的配体。方法用Western blot的方法验证OT3肽(合成的TCRγδ中δ链的CDR3区肽)与PKM2结合,用Western blot及免疫组化法观察PKM2在肿瘤细胞及组织中的表达,用流式细胞术及激光扫描共聚焦显微镜术验证PKM2在肿瘤细胞的定位,用固相化PKM2体外扩增γδT细胞和PKM2刺激γδT细胞分泌IFN-γ的实验检验PKM2的功能。结果 (1)PKM2可与OT3肽结合,肿瘤细胞总蛋白提取物中含有大量PKM2,提示以OT3为探针,从肿瘤细胞总蛋白提取物中钓取到PKM2是合理的,用此方法筛选OT3结合蛋白的方法是可行的;(2)PKM2普遍表达于肿瘤细胞及组织,但不表达于肿瘤细胞膜;(3)PKM2在体外不能扩增γδT细胞,无刺激γδT细胞产生IFN-γ的功能。结论 PKM2不表达于肿瘤细胞表面,对γδT细胞不具有刺激活化的功能,PKM2不具备TCRγδ的配体特性。

Objective To check whether pyruvate kinase isoenzyme type M2（PKM2） is the TCRγδ＇s ligand.Methods By Western blot,the binding of OT3 peptide（the CDR3 region of the δ chain in TCRγδ） with PKM2 was analyzed;with Western blot and immunohistochemistry,the expression of PKM2 in tumor cells and tissues was observed;by flow cytometry and confocal method,PKM2＇s iocation in the tumor cell was tested.The test of immobilized PKM2 expanding γδT cells in vitro and the detection of IFN-γ secreted by PKM2-stimulated γδT cells were used to observe the function of PKM2.Results（1）The binding of PKM2 with OT3 peptide and containing a large number of PKM2 in tumor cells hints that the catching of PKM2 by OT3 peptide from total protein of tumor cells extracts is reasonable and that finding OT3 binding proteins by this method is feasible;（2）PKM2 is extensively expressed on tumor cells and tissues,but not expressed on the tumor cell membrane;（3）PKM2 neither expands γδT cells nor stimulates the secretion of IFN-γ from γδT cells in vitro.Conclusion PKM2 is not expressed on the surface of tumor cells,and can＇t stimulate activation of γδT cells.PKM2 does not have properties to be TCRγδ ligand.