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【目的】构建携带加强型黄色荧光素蛋白 (EYFP)和人类血管内皮生长因子 (VEGF)的双基因真核表达载体pIRES EYFP/VEGF12 1,用EYFP作标记基因 ,研究外源性VEGF12 1基因在大鼠原代培养肝细胞转染表达 ,为实时监测VEGF12 1基因修饰肝细胞移植后状态提供基础。【方法】将 pcDNA3VEGF12 1中VEGF12 1定向克隆到 pIRES EYFP ,构建重组质粒 pIRES EYFP/VEGF12 1,经酶切、PCR扩增和部分DNA序列分析证实后 ,转染大鼠原代培养肝细胞 ,用荧光显微镜等观察转染表达。【结果】酶切、PCR扩增和部分DNA序列分析等证明pIRES EYFP/VEGF12 1质粒成功构建 ,转染大鼠原代培养肝细胞后 ,可以通过荧光显微镜观察到黄绿色荧光。【结论】构建了 pIRES EYFP/VEGF12 1质粒 ,为实时监测外源性VEGF12 1基因转染表达和VEGF基因修饰肝细胞的基因治疗奠定基础。
【Objective】 To construct eukaryotic expression vector pIRES EYFP / VEGF121 carrying enhanced yellow fluorescent protein (EYFP) and human vascular endothelial growth factor (VEGF), and to use EYFP as a marker gene to study the expression of exogenous VEGF12 1 Transfection and expression of rat primary cultured hepatocytes provide the basis for real-time monitoring of the status of VEGF121-modified hepatocytes after transplantation. 【Method】 VEGF12 1 of pcDNA3VEGF12 1 was cloned into pIRES EYFP to construct recombinant plasmid pIRES EYFP / VEGF12 1. After confirmed by restriction enzyme digestion, PCR amplification and partial DNA sequence analysis, primary cultured hepatocytes were transfected with Fluorescence microscopy observation transfection expression. 【Results】 The recombinant plasmid pIRES EYFP / VEGF12 1 was successfully constructed and confirmed by enzyme digestion, PCR amplification and partial DNA sequence analysis. The yellow-green fluorescence was observed by fluorescence microscopy after transfection of rat primary cultured hepatocytes. 【Conclusion】 The pIRES EYFP / VEGF12 1 plasmid was constructed and laid the foundation for the real-time monitoring of gene expression of exogenous VEGF12 1 gene and VEGF gene modified hepatocytes.