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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。
OBJECTIVE: To investigate the effect of introduction of exogenous p16 cDNA eukaryotic expression vector on the biological behavior of human primary hepatocellular carcinoma cell line SMMC-7721 and its molecular mechanism. METHODS: The eukaryotic expression vector of exogenous p16 gene was constructed and transfected into SMMC-772 1 cells. After G4 18 selection and amplification, a stable expression strain was obtained and identified by RT-PCR and immunocytochemistry. Through growth curves, flow cytometry, immunocytochemistry and Western-Blot experimental methods, the biological behavior of tumor cells after transfection and the cell cycle regulatory factors CDK4, CyclinD1 and pRb were observed. RESULTS: The integration and expression of exogenous p16 gene was observed in SMMC-7721 cells transfected with exogenous p16 gene. The growth rate of the cells was significantly slowed and the doubling time was significantly prolonged. G1 phase cells were significantly more than those before transfection (P < 0). .0 5) Immunocytochemistry showed that exogenous p16 expression down-regulated the expression of CDK4 and cylinD1 (P < 0.05), and Western-Blot suggested that the expression of phosphorylated pRb in the cells was significantly decreased after transfection of exogenous p16 gene. . Conclusion: The exogenous p16 gene can be stably expressed in human hepatocellular carcinoma cell line SMMC-772 1 and arrest cells in G1 phase, inhibiting cell growth. The mechanism may be related to the down-regulation of CDK4, cyclinD1 expression and inhibition of pRb phosphorylation.