VEGF真核表达载体构建及其转染骨髓间充质干细胞的表达

摘要

Objective To construct rat VEGF164 eukaryotic expression vector pcDNA3. 1( - )/VEGF164, and to examine the exogenous protein expression in transfected bone marrow mesenchymal stem cells for gene therapy in lung injury. Methods VEGF164 eukaryotic expression vector was constructed and identified with recombinant DNA technology. Vector pcDNA3. 1( - )/VEGF164, pcDNA3. 1( - ) and Lipofectamine 2000TM were transfected into rat marrow mesechymal stem cells of plasmid group, empty plasmid group and liposome group, respectively , with liposome - mediated technique. In vitro VEGF expression was assessed by immunohistochemistry and Western blot. Results The sequencing of pcDNA3. 1( - )/VEGF164 was completely correct. Scattered yellow granules was only observed in pcDNA3.1( - )/VEGF164 - transfected MSCs by immunohistchemistry. Furthermore, Western blot showed significantly higher level of VEGF in cytolytic product in plasmid group than those in the other two groups. Conclusion Eukaryotic expressing vector of pcDNA3. 1( - )/VEGF164 has been successfully constructed and transfected into rat bone marrow mesenchymal stem cells with effective expression of target gene and protein, providing a pontential gene therapy for lung injury.%目的 构建大鼠血管内皮细胞生长因子164(vascular endothelial growth factor,VEGF164)真核表达载体pcDNA3.1(-)/VEGF164,观察大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)转染pcDNA3.1(-)/VEGF164基因后,外源性基因VEGF在蛋白水平的表达,为其在治疗肺组织损伤中的应用奠定基础.方法 应用DNA重组技术构建VEGF164真核表达载体.采用脂质体介导的转染方法将pcDNA3.1(-)/VEGF164转染大鼠MSCs,并设立空质粒对照组(转染pcDNA3.1空载体)、脂质体对照组,用细胞免疫组化和Western blot检测大鼠MSCs 中VEGF164蛋白的表达情况.结果 重组质粒pcDNA3.1(-)/VEGF164测序结果与基因库(GENEBANK)中序列一致.细胞免疫组化检测显示pcDNA3.1(-)/VEGF164转染组MSCs中可见散在黄色颗粒,而空质粒对照组和脂质体对照组均未见明显黄色颗粒.Western blot检测pcDNA3.1(-)/VEGF164转染MSCs 72 h后,细胞裂解产物中VEGF含量明显高于空质粒对照组和脂质体对照组.结论 成功构建了大鼠VEGF164真核表达载体,实现VEGF164基因在大鼠MSCs的成功转染,并有外源性基因及蛋白的表达,具有促进肺组织损伤修复的应用前景.