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目的:克隆AFP启动子和p16基因全长cDNA,构建在肝癌细胞中定向表达p16基因的腺病毒载体,研究p16基因表达对肝癌细胞的影响。方法:采用分子生物学技术手段,克隆AFP启动子和p16基因全长cDNA,将其插入腺病毒载体质粒中,并在293细胞中重组出携带p16基因的腺病毒AdAFP-p16。WesternBlot检测p16基因在肝癌细胞中的特异性表达。细胞病变效应(CPE)观察p16基因表达对肝癌细胞生长的抑制作用。结果:AdAFP-p16能够介导p16基因在肝癌细胞中特异性表达,而对正常细胞中p16的表达没有影响;p16基因表达能够特异性抑制肝癌细胞的生长。结论:采用AFP启动子可以实现p16基因的肝癌细胞中定向而特异性表达,提高基因表达产物在肿瘤局部的浓度,有利于提高治疗效果。
OBJECTIVE: To clone full-length cDNA of AFP promoter and p16 gene and construct adenovirus vector targeting p16 gene in hepatocellular carcinoma cells to study the effect of p16 gene expression on hepatoma cells. Methods: AFP promoter and full length cDNA of p16 gene were cloned by molecular biology techniques. The full length cDNA of AFP promoter and p16 gene was cloned into the adenovirus vector and recombinant adenovirus AdAFP-p16 carrying p16 gene was recombined in 293 cells. Western blot was used to detect the specific expression of p16 gene in hepatoma cells. Cytopathic effect (CPE) to observe the inhibition of p16 gene expression on the growth of hepatocellular carcinoma cells. Results: AdAFP-p16 could mediate the specific expression of p16 gene in hepatoma cells and had no effect on the expression of p16 in normal cells. The expression of p16 gene could specifically inhibit the growth of hepatocellular carcinoma cells. Conclusion: The AFP promoter can be used to achieve specific and directional expression of p16 gene in hepatoma cells, and to increase the concentration of gene expression products in the tumor site, which is beneficial to improve the therapeutic effect.