论文部分内容阅读
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。
Objective: To investigate the effects of adenovirus E1A on apoptosis in rat alveolar epithelial cells (CCL149) and human lung adenocarcinoma cells (A549) induced by TNF-α. Methods: The Pneo-E1A plasmids containing the complete coding region of adenovirus E1A gene were transfected into CCL149 and A549 cells, respectively, and the resistant cell clones were screened by G418. The single cell clones were screened by RT-PCR and immunohistochemistry. Positive clones of E1A gene stably transfected were identified, and the control plasmid transfected cell clones were stimulated with the TNF-α induced apoptosis factor, detected by Hoechest fluorescent staining and flow cytometry combined with Annexin V-FITC labeling Apoptosis. Results: The apoptotic rate of CCL149 cells (C-E1A +) transfected with E1A gene was (2.63 ± 0.8)% and (25.38 ± 0.9)%, respectively, before and after treatment with 30μg / (0.62 ± 0.3)% and (6.08 ± 0.2)% of the transfected cells (C-E1A-), the difference was statistically significant (P <0.01) (5.12 ± 0.5)% and (19.82 ± 1.6)%, respectively, were significantly higher in A549 cells (A-E1A +) treated with 30μg / (2.02 ± 0.7)% before treatment and (9.15 ± 1.2)% after treatment, respectively. The difference between the two groups was statistically significant (P <0.01). Conclusion: E1A protein can increase the sensitivity of cells to apoptosis and up-regulate the apoptosis of cells induced by TNF-α.