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目的观察外源性p16ink4a/hRb1基因联合导入骨肉瘤细胞后,对其细胞周期的协同调控作用。方法利用本室构建的pIRES-p16ink4a-hRb1、pIRES-p16ink4a与pIRES-hRb1质粒,脂质体介导转染p16缺失,hRb1表达阳性的骨肉瘤细胞株MC-63,G418筛选获得抗性克隆,通过逆转录-聚合酶链反应(RT-PCR)和Western blot半定量分析外源基因表达；Sub G_1法流式细胞术分析细胞周期特异性和凋亡率。噻唑蓝(MTT)比色法与细胞生长曲线观察细胞增殖情况。结果外源基因在靶细胞mRNA和蛋白水平分别有独立表达。与对照组比,所有外源性基因导入组细胞周期均显著阻滞在G_1期(P＜0．01)并存在凋亡,且双基因导入组凋亡率分别比单基因组高4．04和6．94倍(P＜0．01)；所有外源性基因导入组各时间点细胞生长抑制率均显著上升,且双基因导入组细胞高于单基因组(P＜0．01)。结论p16ink4a/hRb1联合导入骨肉瘤细胞后,干扰了p16ink4a-Cy- clinD1/CDK-hRb1负反馈循环,比单基因导入更能抑制肿瘤细胞生长和杀灭肿瘤细胞。
Objective To observe the synergistic effect of exogenous p16INK4a / hRb1 gene on osteosarcoma cell line after its induction. Methods The pIRES-p16ink4a-hRb1, pIRES-p16ink4a and pIRES-hRb1 plasmids were transfected into the osteosarcoma cell lines MC-63 and G418 with liposome-mediated transfection of p16 and screened by hRb1. Exogenous gene expression was semi-quantitatively analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Cell cycle specificity and apoptosis rate were analyzed by Sub G-1 flow cytometry. MTT assay and cell growth curve were used to observe cell proliferation. Results The exogenous genes were independently expressed in target cells at mRNA and protein levels respectively. Compared with the control group, the cell cycle of all the exogenous gene transfection groups were significantly arrested in the G_1 phase (P <0.01) and there was apoptosis, and the apoptosis rate in the double gene transfection group was 4.04 and 6.94-fold (P <0.01). The inhibitory rates of all the exogenous gene transfection groups at various time points increased significantly, and the number of cells transfected with the double gene group was higher than that of the single gene group (P <0.01). Conclusion The combination of p16INK4a / hRb1 and osteosarcoma cells can interfere with the negative feedback loop of p16ink4a-CyclKd1 / CDK-hRb1, which can inhibit the growth of tumor cells and kill the tumor cells.