腺病毒介导p16基因的表达及其对肺癌细胞的周期阻滞作用

p16 Gene Expression Mediated by Adenovirus and Its Effect on Cell Cycle Arrest

目的:以增殖缺陷型腺病毒载体介导p16基因感染人肺腺癌细胞系,观察并鉴定该基因在细胞中的表达及其对细 胞周期和细胞凋亡的影响。方法:采用293细胞内同源重组的方法,制备重组腺病毒Ad5-p16,感染肺腺癌细胞SPC-A1;免疫 组化方法鉴定P16蛋白的表达;台盼蓝染色计数活细胞数,绘制细胞生长曲线;流式细胞术(FCM)分析细胞周期及细胞凋亡 的变化。结果:以MOI=10的重组增殖缺陷型腺病毒Ad5-p16感染SPC-A1细胞48 h后,P16蛋白表达的阳性细胞比率为 71％;以MOI=100感染SPC-A1细胞后第2天,细胞开始出现生长抑制及细胞病变;FCM分析发现细胞在感染腺病毒后出现 细胞周期阻滞和细胞凋亡。结论:以腺病毒为载体介导p16基因在SPC-A1细胞内表达,可发挥抑制细胞生长、诱发细胞凋亡 的作用。本项基因治疗策略以直接调控细胞周期的抑癌基因为靶向治疗基因,为肺癌基因治疗提供了可靠的理论依据。

Objective: To investigate the p16 gene expression in lung cancer cell line introduced by adenovirus, and to observe the effect of p16 gene on cell cycle arrest. Methods: The replication-deficient adenovirus Ad5-pl6 was homolo gously recombined in 293 cells and used to infect the lung cancer cell line SPC-A1. The expression of P16 protein was i dentified by immunohistochemistry. Trypan blue staining was used to count the alive cells and to draw a cellular growth curve. The changes of cell cycle and apoptosis were analyzed by flow cytometry (FCM). Results: After 48 hours since the SPC-A1 cells were infected by the replication-deficient adenovirus Ad5-p16 at MOI = 10, the positive rate of the cells for P16 expression was 71%. The cellular growth inhibition and cytopathic effects were present when the cells were infected with Ad5-p16 at MOI = 100. The cell cycle arrest and the cell apoptosis were found by FCM. Conclusion: The expression of p16 gene in SPC-A1 lung cancer cells induced by the replication-deficient adenovirus may result in the inhibition of cell growth and the introduction of cell apoptosis. The strategy targeted to the tumor suppressor gene that regulates the cell cycle directly, obtained satisfactory results, and provided a reliable theory for lung cancer gene therapy.