摘要
目的探讨腺病毒早表达蛋白1A(E1A)对脂多糖、肿瘤坏死因子α(TNF-α)诱导的大鼠肺泡上皮细胞炎症介质细胞间黏附分子1(ICAM-1)表达的影响及其机制。方法致炎因素脂多糖和 TNF-α作用于稳定表达 E1A 的大鼠肺泡卜皮细胞、对照质粒转染细胞和正常大鼠肺泡上皮细胞,采用流式单抗和 RT-PCR 法分析 ICAM-1蛋白水平和 mRNA 水平的表达情况;转录因子报告系统和凝胶电泳迁移变动分析(EMSA)研究核因子(NF-κB)、活化蛋白1与 ICAM-1基因上游调控元件结合的情况。结果 (1)与对照质粒组和正常细胞组相比,E1A 阳性组细胞经10 mg/L 脂多糖和10 pg/L TNF-α刺激后,ICAM-1蛋白表达(任意荧光强度)为109±15和185±20,比对照质粒转染组细胞(60±13,86±22)和正常 CCL149细胞(61±20,89±12)明显升高(F 值分别为14.46、73.64,P 均<0.01);(2)RT-PCR 显示 E1A 阳性组细胞 ICAM-1 mRNA 的表达在脂多糖、TNF-α刺激后3h和6h 均比对照质粒转染组细胞明显增高;(3)转录因子荧光素酶报道系统及 EMSA 结果显示,E1A组在脂多糖、TNF-α作用前后,细胞核内 NF-κB与 ICAM-1基因上游调控序列结合形成的阻滞条带强度均明显高于埘照组;而活化蛋白1与 ICAM-1基因上游调控序列特异性结合形成的阻滞条带在E1A 阳性组和对照组在刺激因素作用前后比较无明显差异;(4)在脂多糖作用后,NF-κB抑制剂 N-甲苯磺基-L-苯乙胺酰氯甲基酮(TPCK)可使 E1A 组 ICAM-1蛋白表达强度下降(109±15,50±10);TNF-α作用后 E1A 组 ICAM-1蛋白表达强度也明显下降(185±20,55±13),TPCK 处理前后比较,差异有统计学意义(t 值分别为8.4、12.2,P 值分别为0.01和0.00)。结论 E1A 可明显上调炎症介质ICAM-1的表达,上调转录因子 NF-κB、活化蛋白1的活性;E1A 上调 ICAM-1的表达主要通过 NF-κB实现。
Objective The relationship between latent adenvorius infection and airway inflammation has not been well documented. The aim of this study is to illustrate the roles of adenovirus E1A protein on the inflammation mediator expression in response to lipopolysaccharide and tumor necrosis factor α（TNF-α） in rat alveolar epithelial cells. Methods An eukaryotic expression vector for expressing adenovirus E1A protein was constructed and transfected into CCL149 cells. Cells stably expressing E1A protein were selected by G418 resistance. The inflammatory mediator intercellular adhesion molecule-1 （ ICAM-1 ） expression in response to lipopolysaccharide and TNF-α was compared between adenovirus E1A-positive clones, control clones and CCL149 cells. Messenger RNA of ICAM-1 was measured by RT-PCR,and proteins quantified by flow cytometry. The nuclear factor κB （NF-κB） and activator protein 1 （ AP-1 ） activity were measured by LUC report system and electrophoretic mobility shift assay （EMSA）. Results ICAM-1 messenger RNA and protein were increased in E1A-positive cells exposed to 10 ng/ml TNF-α and 10 μg/ml lipopolysaccharide. The luciferase activity drived by NF-κB and AP-1 elements were increased in E1A-positive cells compared with control with or without lipopolysaccharide and TNF-α stimulation. EMSA showed that only NF-κB activity increased in E1A-positive cells. This increase was not observed in AP1 element drived EMSA.Conclusions The results indicate that EIA upregulates ICAM-1 expression induced by lipopolysaccharide and TNF-α in rat alveolar epithelial cells. E1A enhances the expression of inflammatory mediator by triggering NF-κB activity. It is suggested that E1A amplified inflammatory response may contribute to the pathogenesis of chronic obstructive pulmonary disease.
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2007年第8期582-587,共6页
Chinese Journal of Tuberculosis and Respiratory Diseases
基金
广东省自然科学基金团队项目(05200239)
关键词
腺病毒科
病毒潜伏期
即早蛋白质类
炎症
Adenoviridae
Virus latency
Immediate-early proteins
Inflammation
