论文部分内容阅读
目的建立肿瘤坏死因子α(TNF-α)诱导支气管上皮(16HBE)细胞炎症模型,探讨谷胱甘肽S转移酶mu5(GSTM5)在炎症诱导的氧化应激损伤中的作用。方法以TNF-α(10 ng/m L)刺激16HBE细胞,运用真核细胞表达载体技术,增强GSTM5基因表达,比色法检测细胞内丙二醛(MDA)及总抗氧化能力(T-AOC);MTT法检测细胞存活率;RT-PCR法检测NADPH氧化酶(NOX)家族NOX1、NOX2、NOX3、NOX4、NOX5、双重氧化酶1(DUOX1)、DUOX2相关基因转录水平;蛋白质印迹法检测NOX1和NOX2蛋白表达水平。结果 TNF-α刺激16HBE细胞后,细胞内MDA水平增高,T-AOC能力减低,细胞存活率明显降低。预转染GSTM5质粒可有效降低TNF-α诱导的细胞内MDA的增高(P<0.05),显著增强T-AOC能力(P<0.05),同时增加16HBE细胞存活率(P<0.05)。GSTM5质粒组NOX1、NOX2基因转录强度以及蛋白表达水平与TNF-α组及阴性对照组相比显著降低(P均<0.05),而NOX3、NOX4、NOX5、DUOX1、DUOX2基因表达强度比较,差异无统计学意义(P均>0.05)。结论增强GSTM5表达对炎症诱发的人支气管上皮细胞氧化应激损伤有保护性调节作用,其机制与下调NOX1、NOX2基因表达密切相关。
Objective To establish a model of bronchial epithelial (16HBE) cell inflammation induced by tumor necrosis factor α (TNF-α) and to explore the role of glutathione S-transferase mu5 (GSTM5) in inflammation-induced oxidative stress injury. Methods 16HBE cells were stimulated with TNF-α (10 ng / m L), and the expression of GSTM5 gene was enhanced by eukaryotic expression vector. The contents of malondialdehyde (MDA) and total antioxidant capacity (T-AOC ). The cell viability was detected by MTT assay. The transcription levels of NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2 were detected by RT-PCR and NOX1 And NOX2 protein expression levels. Results After stimulated by TNF-α for 16HBE cells, the level of intracellular MDA increased, T-AOC decreased and the cell survival rate decreased significantly. The transfection of GSTM5 plasmid could effectively decrease the intracellular MDA level induced by TNF-α (P <0.05), enhance the ability of T-AOC (P <0.05) and increase the survival rate of 16HBE cells (P <0.05). Compared with TNF-α group and negative control group, the transcriptional intensity and protein expression of NOX1 and NOX2 in GSTM5 plasmid group were significantly decreased (all P <0.05), while the expression intensity of NOX3, NOX4, NOX5, DUOX1 and DUOX2 in GSTM5 plasmid group was significantly lower than that in TNF- Statistical significance (P> 0.05). Conclusion Enhanced expression of GSTM5 may play a protective role in oxidative stress injury induced by inflammation in human bronchial epithelial cells. The mechanism is related to the down-regulation of NOX1 and NOX2 gene expression.