转录因子Klf2和Nrf2/Bach1调控γ-谷氨酰半胱氨酸合酶在气道上皮细胞中的作用  

Regulation of Transcription Factors Klf2 and Nrf2/Bach1 on Gamma-Glutamylcysteine Synthetase in Bronchial Epithelial Cells

作　　者：黄畅宇[1,2] 戴爱国 

机构地区：[1]南华大学研究生院,湖南衡阳421001 [2]湖南省老年医院老年医学研究所呼吸疾病研究室,湖南长沙410016

出　　处：《中国呼吸与危重监护杂志》2012年第1期23-27,共5页Chinese Journal of Respiratory and Critical Care Medicine

基　　金：湖南省科技计划重点项目(编号:2007FJ3016;2008FJ4206);湖南省医药卫生科研计划项目(编号:B2009068)

摘　　要：目的通过研究锌指Krüppel样转录因子2(Klf2)及红系衍生核因子相关因子2(Nrf2)/BTB-CNC异体同源体1(Bach1)在经香烟烟雾提取物(CSE)刺激的大鼠气道上皮细胞中的表达,了解其感受氧化应激对靶基因γ-谷氨酰半胱氨酸合酶(γ-GCS)的调控作用。方法采用酶消化法提取大鼠原代气管-支气管上皮细胞,并用10%CSE干预细胞6 h,收集细胞,双酶法检测γ-GCS活性;通过免疫细胞化学法观察Nrf2/Bach1的核转位;采用Western blot和RT-PCR技术研究细胞中Klf2、Nrf2、Bach1、γ-GCS的蛋白及mRNA表达。结果大鼠气管-支气管上皮细胞经CSE诱导后其γ-GCS活性明显增高。免疫细胞化学结果显示Nrf2经CSE刺激后出现由核外向核内的转位,Bach1经CSE刺激后出现由核内向核外的转位。Western blot结果显示CSE处理组中Klf2、Nrf2、Bach1、γ-GCS蛋白水平较对照组显著升高(P<0.05);RT-PCR结果显示CSE处理组中Klf2、Nrf2、Bach1、γ-GCS mRNA水平较对照组显著升高(P<0.05)。直线相关分析表明γ-GCS蛋白表达与Klf2、Nrf2、Bach1呈正相关(P<0.05)。结论 CSE可能通过转录因子Klf2、Nrf2、Bach1调节γ-GCS的表达。Objective To investigate the expression of transcriptional factors zinc finger Krüppel like transcription factor 2（Klf2） and NF-E2 related factor 2（Nrf2）/BTB CNC homology 1（Bach1） in rat bronchial epithelial cells stimulated by cigarette smoke extract（CSE）,and explore the regulating mechanism of γ-glutamylcysteine synthetase（γ-GCS） expression in the oxidative condition.Methods Rat bronchial epithelial cells were harvested using enzyme digestion method,and intervened by 10% CSE for 6 hours.Then γ-GCS activity was detected by two enzymes method,and the nuclear transfer of Nrf2/Bach1 in cells was detected by immunohistochemistry.Western blot and reverse transcription-polymerase chain reaction（RT-PCR） techniques were used for detecting the protein and mRNA expressions of Klf2,Nrf2,Bach1,and γ-GCS in the cells.Results The γ-GCS activity was elevated in the CSE group.Immunohistochemical results showed that Nrf2 translocated from cytoplasm to nucleus in response to stimulation by CSE.On the contrary,Bach1 translocated from nucleus to cytoplasm in the same condition.Western blot results showed that protein levels of Klf2,Nrf2,Bach1,and γ-GCS were higher in the CSE group than those in the control group（P〈0.05）.RT-PCR results were the same as the Western blot results（P〈0.05）.Linear correlation analysis showed that γ-GCS expression was positively correlated with Klf2,Nrf2,and Bach1（P〈0.05）.Conclusion CSE might regulate the expression of γ-GCS through the transcription factors of Klf2,Nrf2,and Bach1.

关 键 词：香烟烟雾提取物 锌指Krüppel样转录因子2 红系衍生核因子相关因子2 BTB-CNC异体同源体1 Γ-谷氨酰半胱氨酸合酶 

分 类 号：R363[医药卫生—病理学]