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目的:研究腺病毒Ela基因与P16抑癌基因协同对肝癌SMMC-7721细胞凋亡和增殖的影响,探索肿瘤基因治疗新模式。方法:构建Ela基因真核表达质粒pDC315-E1a和携带P16的重组病毒AdCMV-P16,RT-PCR和免疫荧光标记法检测pDC315-E1a质粒转染或AdCMV-P16病毒感染后SMMC-7721细胞中P16和Ela的表达。建立裸鼠SMMC-7721细胞移植瘤模型,pDC315-E1a和AdCMV-P16单独或联合治疗,观察其对移植瘤生长的抑制作用,免疫组化和TUNEL法分别检测移植瘤组织中P16、E1a蛋白的表达和移植瘤细胞的凋亡。结果:SMMC-7721细胞感染AdCMV-P16或转染pDC315-E1a后,P16、Ela mRNA和蛋白水平均呈阳性表达。与空白对照组相比,AdCMV-P16治疗组移植瘤细胞凋亡率为(14.3±2.5)%(P<0.01),抑瘤率为36.1%(P<0.01);pDC315-E1a治疗组抑瘤率为17.1%(P>0.05),移植瘤细胞凋亡率为(8.5±2.9)%(P<0.01);AdCMV-P16联合pDC315-E1a治疗组移植瘤细胞凋亡率为(27.3±6.3)%(P<0.01),抑瘤率达57.2%(P<0.01)。结论:腺病毒Ela基因能够增强P16基因对肝癌SMMC-7721细胞移植瘤的抑制作用,促进移植瘤细胞的凋亡,增强P16基因治疗的效果。
Objective: To study the effect of adenovirus Ela gene and P16 tumor suppressor gene on the apoptosis and proliferation of hepatocellular carcinoma SMMC-7721 cells and to explore a new model of tumor gene therapy. Methods: The eukaryotic expression plasmid pDC315-E1a of Ela gene and the recombinant virus AdCMV-P16 carrying P16 were constructed. The expression of P16 in SMMC-7721 cells transfected with pDC315-E1a or AdCMV-P16 was detected by RT-PCR and immunofluorescence staining And Ela expression. The nude mouse SMMC-7721 xenografts model was established. The pDC315-E1a and AdCMV-P16 alone or in combination therapy were used to observe the inhibitory effect on the growth of the transplanted tumor. Immunohistochemistry and TUNEL were used to detect the expression of P16 and E1a Expression and Apoptosis of Transplanted Tumor Cells. Results: The expression of P16 and Ela mRNA and protein were all positive in SMMC-7721 cells infected with AdCMV-P16 or transfected with pDC315-E1a. Compared with the blank control group, the apoptosis rate of the tumor cells in AdCMV-P16 group was (14.3 ± 2.5)% (P <0.01), and the inhibition rate was 36.1% (P <0.01) The apoptotic rate of transplanted tumor cells was (8.5 ± 2.9)% (P <0.01), and the apoptotic rate of transplanted tumor cells was (27.3 ± 6.3) days after treatment with AdCMV-P16 and pDC315-E1a % (P <0.01), and the inhibition rate was 57.2% (P <0.01). Conclusion: The adenovirus Ela gene can enhance the inhibitory effect of P16 gene on the transplanted tumor of hepatocellular carcinoma SMMC-7721 cells and promote the apoptosis of transplanted tumor cells and enhance the effect of P16 gene therapy.