氧化应激对腺病毒E1A蛋白活化NF-κB转录活性的影响及其机制研究  

The oxidative stress enhanced adenoviral E1A-involved transactivation of nuclear factor-κB

作　　者：陈娟[1] 方怡[2] 付欣[3] 李冰[3] 张锦[1] 冉丕鑫[4] 

机构地区：[1]宁夏医科大学附属医院呼吸内科,银川750004 [2]广州军区总医院,广东广州510010 [3]广州医学院实验医学研究中心,广东广州510182 [4]广州医学院第一附属医院广州呼吸病研究所,广东广州510120

出　　处：《中国应用生理学杂志》2010年第4期395-398,共4页Chinese Journal of Applied Physiology

基　　金：国家自然科学基金青年科学基金项目(30800501);广东省自然科学基金团队项目(05200239);宁夏自然科学基金项目(NZ10137)

摘　　要：目的:探讨腺病毒E1A蛋白对细胞内抗氧化物质谷胱甘肽(GSH)水平的影响及氧化应激对腺病毒E1A蛋白介导的核因子-κB(NF-κB)转录活化的影响。方法:构建稳定表达E1A蛋白的大鼠肺泡上皮细胞(E1A组)及对照质粒转染细胞(对照组),每组5×105个细胞,试验重复3次。采用H2O2刺激细胞,检测细胞内GSH水平。脂多糖(LPS)和肿瘤坏死因子-α(TNF-α)进行刺激,丁胱亚磺酰亚胺(BSO)进行干预,Western blot法检测NF-κB和AP-1蛋白的表达。结果:E1A+细胞内GSH基础水平与E1A-比较无显著差异,但在H2O2作用后没有诱导出GSH含量的上升,表现为下降而低平的趋势,去除氧化剂后,仍低于正常水平。E1A-细胞在氧化剂的作用后呈明显的上升趋势,去除氧化剂后仍高于基础水平。细胞内NF-κB蛋白表达(积分吸光度值):刺激前E1A+细胞分别为79.3±4.6和80.3±3.8,在LPS和TNF-α刺激后分别为81.8±3.9～89.9±1.6和94.1±1.9～99.8±1.6,均明显高于对照组(刺激前分别为68.3±3.8和69.4±4.3,刺激后分别为70.1±2.8～80.8±3.6和73.4±4.9～83.2±6.7)。给予BSO预处理后再用LPS和TNF-α刺激,E1A-细胞NF-κB蛋白表达的积分吸光度值(1.22±0.16和1.75±0.13)与LPS或TNF-α单独作用组(1.25±0.18和1.69±0.19)无明显差别;E1A+细胞NF-κB蛋白表达的积分吸光度值(1.75±0.10和2.26±0.21)明显高于LPS或TNF-α单独作用组(1.35±0.12和1.80±0.14)。结论:腺病毒潜伏感染持续表达E1A蛋白可以影响细胞GSH,降低细胞对氧化应激的耐受性,并可能通过此机制造成NF-κB异常转录活化,而GSH的降低又进一步放大了E1A介导的NF-κB转录活化作用。Objective：The relationship between latent adenovirus infection and airway inflammation have not been well documented. The aim of this study is to illustrate the roles of adenovirus E1A protein on the level of glutathione（GSH） in response to oxidative stress and the effect of the oxidant/antioxidant inbalance upon the transactivation of NF-κB triggered by E1A protein. Methods： Rat alveolar epithelial cell stably expressing adenoviral E1A or control plasmid were developed. For isolation of nuclear extracts,5×105 cells were plated and grown overnight in 60 mm dishes.Experiments were repeated three times.The cell model of stably expressing adenoviral E1A was stimulated by H2O2. The level of GSH were measured. E1A positived clone was stimulated by LPS or TNF-α and treated with L-Buthionine-sulfoximine（BSO） The expression of NF-κB was measured by Western blot.Differences between groups were assessed for significance by Student’ t test;multiple comparisons by the one-way ANOVA. Results： There is no difference of GSH level without stimulation between E1A-positive clones and E1A-ne-gative clones. For E1A-positive clones,the level of GSH did not increase in response to H2O2 as E1A-negative clones.The quantitation by densitometry of the NF-κB expression in E1A-positive clones were （79.3±4.6）,（80.3±3.8） respectively without treatment and were （81.8±3.9）～（89.9±1.6） and （94.1±1.9）～（99.8±1.6） respectively under LPS or TNF-α stimulation,which were significantly higher than that of the control group（（68.3±3.8）,（69.4±4.3） respectively without stimulation and （70.1±2.8）～（80.8±3.6）,（ 73.4±4.9）～（83.2±6.7） respectively under stimulation.The quantitation by densitometry of the NF-κB expression in E1A-negative clones were （1.25±0.18） and （1.69±0.19） respectively under LPS and TNF-α-stimulation and （1.22±0.16） and （1.75±0.13） respectively upon treatment for LPS and TNF-α with BSO preincubation.There did not show difference u

关 键 词：病毒潜伏期 氧化应激 转录因子 

分 类 号：R56[医药卫生—呼吸系统]