中国呼吸与危重监护杂志

中国呼吸与危重监护杂志

肿瘤坏死因子-α 可诱导 GSTM5 核转位

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目的构建稳定表达谷胱甘肽 S 转移酶 mu5(GSTM5)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-α 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-α 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。

ObjectiveTo establish 16HBE cell lines stably expressing glutathione S-transferase mu 5 (GSTM5) gene, and explore the mechanism of GSTM5 nuclear translocation. MethodsRecombinant 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-α; 10 ng/ml) for 0.5 hour. ResultsLentiviral 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. ConclusionThe 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.

关键词: 谷胱甘肽 S 转移酶 mu 5; 重组慢病毒表达质粒; 16HBE; 核转位; 肿瘤坏死因子-α

Key words: Glutathione S-transferase mu 5; Recombinant lentiviral expression vector; 16HBE; Nuclear translocation; Tumor necrosis factor-α

引用本文: 曾燕婷, 李理, 袁伟锋, 郭振辉, 黄文杰. 肿瘤坏死因子-α 可诱导 GSTM5 核转位. 中国呼吸与危重监护杂志, 2017, 16(5): 451-456. doi: 10.7507/1671-6205.201611031 复制

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