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目的 构建稳定表达谷胱甘肽S转移酶mu5 (GSTMS)的人支气管上皮16HBE细胞株,探讨诱导GSTM5核转位的关键因素.方法 构建表达GSTM5基因的重组慢病毒载体(PLVX-puro-GSTM5-SBP-3*flag-N、PLVX-puro-3*flag-SBP-GSTM5-C)并制备出相应的慢病毒,感染人支气管上皮16HBE细胞后,经嘌呤霉素筛选获得细胞克隆;实时荧光定量PCR和蛋白印迹法(Western blot)分别检测细胞株中GSTM5的mRNA、蛋白表达水平;以肿瘤坏死因子-α (TNF-α;10 ng/ml)刺激稳定转染细胞株(稳转株)0.5 h,共聚焦荧光显微镜检测GSTM5核转位.结果 成功构建稳定表达GSTM5人支气管上皮细胞株(16HBE-GSTM5-SBP-3*flag-N、16HBE-3*flag-SBP-GSTM5-C);荧光共聚焦显微镜显示稳转株在TNF-αt刺激诱导后,GSTM5由胞浆转入胞核,以16HBE-GSTM5-SBP-3*flag-N稳转株更显著.结论 TNF-αt刺激可诱导GSTM5人支气管上皮细胞株中GSTM5发生核转位,可能与其N端含有非经典核定位信号密切相关.“,”Objective To establish 16HBE cell lines stably expressing glutathione S-transferase mu 5 (GSTM5) gene,and explore the mechanism of GSTM5 nuclear translocation.Methods Recombinant lentiviral expression vector containing GSTM5 gene was constructed and lentivirus was produced.After lentivirus infection of 16HBE cells,16HBE-GSTM5 cell lines were obtained by screening with puromycin.Expression of GSTM5 in different cells was examined by RT-qPCR and Western blot.The nuclear translocation of GSTM5 was observed by confocal laser scanning microscope,after the 16HBE-GSTM5 cell lines were treated with tumor necrosis factor-α (TNF-ct;10 ng/ml) for 0.5 hour.Results Len tiviral expression plasmids,PLVX-puro-3*flag-SBP-GSTM5-C and PLVX-puro-GSTM5-SBP-3*flag-N,were constructed and lentiviral particles were successfully packed.After infected with lentivirus and screened by puromycin,two cell lines,16HBE-GSTM5-SBP-3*flag-N and 16HBE-3*flag-SBP-GSTM5-C,were obtained.GSTM5 expression in these two cell lines was significantly higher compared with the control group and parental cells.After treated with TNF-α for 0.5 hour,the nuclear translocation of GSTM5 in 16HBE-GSTM5-SBP-3*flag-N was much more obviously than that in 16HBE-3*flag-SBP-GSTM5-C.Conclusion The N-terminal region of GSTM5 is critical for nuclear translocation induced by TNF-α,which is mediated by a novel and non-classical nuclear localization signal.