肿瘤坏死因子-α可诱导GSTM5核转位

发布时间：2018-05-22 10:55

  本文选题：谷胱甘肽S转移酶mu + 重组慢病毒表达质粒　； 参考：《中国呼吸与危重监护杂志》2017年05期

【摘要】：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;10ng/ml)刺激稳定转染细胞株(稳转株)0.5h,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*flagSBP-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16HBEGSTM5-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。
[Abstract]:Objective to construct a human bronchial epithelial 16HBE cell line stably expressing glutathione S transferase mu5 (GSTM5) and to explore the key factors of inducing GSTM5 nuclear translocation. Methods Recombinant lentivirus vector PLVX-puro-GSTM5-SBP-3 flag-nban-PLVX-puro-3ban-SBP-GSTM5-C) expressing GSTM5 gene was constructed, and the corresponding lentivirus was prepared. Human bronchial epithelial 16HBE cells were infected with PLVX-puro-GSTM5-SBP-3 flag-SBP-GSTM5-C5. The clones were obtained by purine mycin screening. The expression of GSTM5 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot respectively, and the nuclear translocation of GSTM5 was detected by confocal fluorescence microscope (confocal fluorescence microscope) after the tumor necrosis factor- 伪 TNF- 伪 10 ng / ml was stimulated to stably transfect the cell line (stable strain 0. 5 h). Results stable expression of GSTM5 in human bronchial epithelial cell line (16HBE-GSTM5-SBP-3) was successfully constructed, and fluorescent confocal microscopy showed that the stable transformed strain was transferred from cytoplasm to nucleus after TNF- 伪 stimulation. Conclusion TNF- 伪 stimulation can induce nuclear translocation of GSTM5 in GSTM5 human bronchial epithelial cell line, which may be closely related to the presence of non-classical nuclear localization signal in the N-terminal of TNF- 伪.
【作者单位】： 南方医科大学研究生学院;广州军区广州总医院呼吸内科;广州军区广州总医院MICU科;
【基金】：国家自然科学基金面上项目(81370173);国家自然科学基金面上项目(81070003) 广东省老年感染与器官功能支持重点实验室 广州市老年感染与脏器功能支持重点实验室资金
【分类号】：R563.8
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本文编号：1921770