摘要
目的研究白三烯诱导人呼吸道上皮细胞内谷胱甘肽氧化还原状态变化及γ-谷氨酰半胱氨酸合成酶(γ-GCS)的作用。方法将人气道上皮细胞分为对照组和暴露组。暴露组的人气道上皮细胞暴露于白三烯2、4、6、12 h。检测对照组及暴露组在2、4、6、12 h相应指标。测定气道上皮细胞内谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)含量;双酶法测定γ-GCS活性;RT-PCR检测气道上皮细胞中γ-GCS-mRNA表达;Western-blot检测气道上皮细胞核和细胞质中γ-GCS蛋白表达;细胞免疫荧光法检测气道上皮细胞γ-GCS蛋白表达。结果暴露组细胞2、4、6、12 h的GSH含量、GSH/GSSG比值先减少后逐渐增加,4 h最低,6 h达最高,12 h恢复正常;而GSSG含量则呈现相反趋势,与对照组之间差异有统计学意义。暴露组细胞2、4、6 h,γ-GCS活性增强,与对照组比较差异有统计学意义。细胞免疫荧光法检测气道上皮细胞γ-GCS蛋白阳性信号主要分布在胞质中,暴露组呈强阳性表达,而对照组表达较弱,暴露组2、4、6 h时与对照组比较,差异有统计学意义。Western-blot检测结果相似。RT-PCR检测气道上皮细胞中γ-GCS-mRNA表达,暴露组2、4、6 h时较对照组增加,差异有统计学意义。结论白三烯诱导氧化应激影响呼吸道上皮细胞谷胱甘肽氧化还原状态,而呼吸道上皮细胞通过提高γ-GCS活性对抗氧化应激,保持氧化还原稳定的状态。
Objective To investigate the influence of leukotriene on glutathione redox state and γ-glutamylcysteine synthetase （γ-GcS） in cultured human airway epithelial cells. Methods Cultured human airway epithelial cells were divided into two groups, experiment group and control group. Experiment group was exposed to leukotriene for 2, 4, 6 and 12 hours. The GSH and GSSG concentration were measured. Activity of γ-GCS was detected by dual enzymatic method. Expression of γ-GCS was detected by RT-PCR, Western-blot, and immunofluorescence method. Results The GSH concentration and GSH/GSSG ratio were decreased first, but then gradually increased in experimental group with the lowest at 4 h and the highest at 6 h, and returned to be normal at 12 h. The concentration of GSSH showed the opposite trend. Activity of γ-GCS at 2 h, 4 h and 6 h in experiment group was significantly enhanced than that in control group. γ-GCS protein, mainly expressed in cytoplasm, was significantly stronger in experiment group than that in control group at 2 h, 4 h and 6 h. Western blot test had the similar result. Expression of ^-GCS mRNA in experiment group was significantly increased than that in control. Conclusions Leukotriene affected glutathione redox state in cultured human airway eDithelial cells by inducing oxidative stress, which might be antagonized through γ-GCS.
出处
《临床儿科杂志》
CAS
CSCD
北大核心
2012年第1期66-69,共4页
Journal of Clinical Pediatrics
