腺病毒E1A蛋白对大鼠肺泡上皮细胞及人肺腺癌细胞凋亡的影响  

Effect of adenoviral E1A-involved apoptosis of rat alveolar epithelial cells and hunma lung adenocarcinoma epithelial cell line

作　　者：陈娟[1] 张锦[1] 郑西卫[1] 郭园园[1] 付欣[2] 李冰[2] 冉丕鑫[3] 

机构地区：[1]宁夏医科大学附属医院呼吸内科,宁夏银川750004 [2]广州医学院实验医学研究中心 [3]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：《细胞与分子免疫学杂志》2010年第5期423-426,共4页Chinese Journal of Cellular and Molecular Immunology

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);宁夏医科大学附属医院院启动课题(2009)

摘　　要：目的:探讨腺病毒E1A蛋白对大鼠肺泡上皮细胞(CCL149)及人肺腺癌细胞(A549)在致凋亡因素TNF-α诱导下细胞凋亡影响。方法:将含腺病毒E1A基因完全编码区的Pneo-E1A质粒分别转染CCL149、A549细胞,用G418筛选抗性细胞克隆,用RT-PCR、免疫组化方法对单个细胞克隆进行筛选鉴定;将经鉴定确定的稳定转染E1A基因的阳性细胞克隆、对照质粒转染细胞克隆用致凋亡因素TNF-α刺激,用Hoechest荧光染色分析及流式细胞仪结合膜联蛋白V-FITC标记法检测细胞凋亡情况。结果:稳定转染E1A基因的CCL149细胞(C-E1A+)在30μg/LTNF-α作用前、后细胞的凋亡率分别为(2.63±0.8)%和(25.38±0.9)%,明显高于对照质粒转染细胞(C-E1A-)作用前的(0.62±0.3)%和作用后的(6.08±0.2)%,两组相比差别有统计学意义(P<0.01);稳定转染E1A基因的A549细胞(A-E1A+)在30μg/LTNF-α作用前、后细胞的凋亡率分别为(5.12±0.5)%和(19.82±1.6)%,明显高于对照质粒转染细胞(A-E1A-)作用前的(2.02±0.7)%和作用后的(9.15±1.2)%,两组相比差别有统计学意义(P<0.01)。结论:E1A蛋白能够增加细胞对致凋亡因素的敏感性,上调TNF-α诱导下的细胞凋亡。AIM：The relationship between latent adenvorius infection and apoptosis of airway epithelial cell have not been well documented.We want to illustrating the roles of adenovirus E1A protein on the apoptotic alveolar epithelial in response to TNF-α.METHODS：The expression vector for expressing adenovirus E1A protein was transfected into CCL149 and A549 cell respectively.Cell stably expressing E1A protein were selected by G418 resistance.All G418-resistant clones were indentified by RT-PCR and immunocytochemistry.The rate of apoptosis were measured by Hoeschest 33 258 and flow cytometry respectively.The apoptotic rate in response to 30 μg/L TNF-α was compared between E1A-positive clones and control clones both in A549 and CCL149.RESULTS：The rate of apoptosis were （2.63±0.8）%,（25.38±0.9）% respectively in E1A-positive CCL149 cell and （0.62± 0.3）%,（6.08±0.2）% respectively in E1A-negative CCL149 cell.The rate of apoptosis were （2.63±0.8）%,（25.38±0.9）% respectively in E1A-positive A549 cell and （0.62± 0.3）%,（6.08±0.2）% respectively in E1A-negative A549 cell.The rate of apoptosis were increased in E1A-positive cells compared with control with or without TNF-α stimulation.CONCLUSION：E1A sensitizes cell to TNF-α induced apoptosis of A549 cell and CCL149 cell.

关 键 词：腺病毒 潜伏感染 凋亡 

分 类 号：R56[医药卫生—呼吸系统]