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目的 通过将Angiopoietin 1基因转染培养人肝癌细胞SMMC 772 1,建立长期表达Angiopoietin 1基因的肝癌细胞模型。 方法 基因重组构建Angiopoietin 1真核表达质粒 pcDNA3/Angiopoietin 1,经脂质体将 pcDNA3/Angiopoietin 1质粒转染培养人肝癌细胞株SMMC 772 1,G418筛选阳性细胞克隆 ,逆转录 聚合酶链反应 (RT PCR)检测转染后 2 5～ 30d细胞Angiopoietin 1基因mRNA表达水平。结果 ( 1)限制酶切和凝胶电泳证明成功构建表达质粒pcDNA3/Angiopoietin 1;( 2 )通过脂质体Dosper将质粒 pcDNA3/Angiopoietin 1转染SMMC 772 1,经G418筛选后获得阳性细胞克隆 ;( 3)RT PCR证明经脂质体转染的人肝癌细胞株SMMC 772 1可较稳定表达Angiopoi etin 1基因。结论 成功建立稳定表达Angiopoietin 1基因的肝癌细胞株SMMC 772 1/Angiopoi etin 1,为通过在体途径探讨Angiopoietin 1对肿瘤血管生成的调节作用奠定了基础。
Objective To establish an animal model of hepatoma expressing long-term Angiopoietin 1 gene by transfecting Angiopoietin 1 gene into human hepatoma SMMC 772 1. METHODS: Angiopoietin 1 eukaryotic expression plasmid pcDNA3/Angiopoietin was constructed by gene recombination. The pcDNA3/Angiopoietin 1 plasmid was transfected into human hepatoma cell line SMMC 772 1 by liposome. G418 was screened for positive cell clones, and reverse transcriptase polymerase chain reaction (RT). PCR was used to detect the mRNA expression level of Angiopoietin 1 at 25-30 days after transfection. Results (1) Restriction enzyme digestion and gel electrophoresis demonstrated the successful construction of the expression plasmid pcDNA3/Angiopoietin 1; (2) The plasmid pcDNA3/Angiopoietin 1 was transfected into SMMC 772 1 by Lipofectin Dosper and screened with G418 to obtain positive cell clones; (3) RT PCR demonstrated that liposome-transfected human hepatoma cell line SMMC 772 1 can more stably express Angiopoi etin 1 gene. Conclusion The successful establishment of SMMC 772 1/Angiopoi etin 1, a hepatoma cell line that stably expresses Angiopoietin 1 gene, lays the foundation for exploring the role of Angiopoietin 1 in regulating tumor angiogenesis in vivo.