【摘要】 目的:探讨导入外源性p16cDNA真核表达载体对人原发性肝癌细胞系SMMC- 772 1生物学行为的影响及其分子机制。方法:构建外源性p16基因真核表达载体并转染SMMC- 772 1细胞,经G4 18抗性筛选和扩增,得到稳定的表达株,并经RT -PCR及免疫细胞化学鉴定。通过生长曲线,流式细胞术,免疫细胞化学及Western -Blot等实验方法,对转基因后肿瘤细胞生物学行为及细胞周期调控因子CDK4 ,CyclinD1及pRb进行观察。结果:转染外源性p16基因的SMMC- 772 1细胞有外源性p16基因的整合及表达,细胞生长速度明显减慢,倍增时间明显延长,G1期细胞明显多于转基因前(P <0 .0 5 ) ;免疫细胞化学显示外源性p16表达可下调CDK4及cylinD1的表达(P <0 .0 5 ) ,Western -Blot提示转染外源性p16基因后细胞中磷酸化pRb表达明显降低。结论:外源性p16基因导入人肝癌细胞株SMMC -772 1中可稳定表达并使细胞停滞在G1期,抑制细胞生长,其机制可能为下调CDK4、cyclinD1的表达和抑制pRb磷酸化有关。更多还原

【Abstract】 Objective To investigate the effect of exogenous p16 ex pression on b iological behavior of human hepatocellular carcinoma and to explore the mechanis m of the growth suppression of p16 in hepatocarcinoma cell line. Methods pcDNA3.1 (+)-p16 eukaryotic expression plasmid was constructed a nd transfected into SMM C-7721 cell which retains down- regulated p16 gene expression. Integration and e xpression of exogenous p16 gene were confirmed by RT-PCR and immunocytochemistr y . The curve of cell growth rate,flow cytometry,immunocytochemistry and weste rn-blot were used to observe the biological behaviors of p16 gene transfected h epatocellular carcinoma cell and to investigate the effect of exogenous p16 exp ression on CDK4,Cyclin D1 and pRb. Results The growth of the cells transfected with p16 was decreased and the percentage of the cell in G0/G1 phase was increas ed. The expression of cylin D1,CDK4 and phosphorylated pRb were down-regula ted in the cells which transfected with p16 gene. Conclusion Exoge nous p16 gene co uld be expressed in SMMC-7721. It may arrest cell cycle in G0/G1 phase and supp r ess cell growth. The mechanism may be that p16 gene down-regulate the expressio n of cylin D1 and CDK4, and the final result constributes to the inhibition o f pRb phosphorylation.更多还原