VEGF逆转录病毒载体构建及在小鼠骨髓细胞中的表达

摘要

目的构建人VEGF121逆转录病毒表达载体,探讨应用血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)基因治疗骨科缺血性疾患的可行性.方法采用分子克隆技术从pCDⅠ/VEGF121质粒获得hVEGF121cDNA, 并克隆到pLXSN逆转录病毒质粒.经酶切及测序鉴定正确后,包装成为pLXSN/VEGF121重组逆转录病毒, 并进行滴度测定.培养小鼠骨髓基质细胞,用重组病毒感染小鼠骨髓基质细胞.经筛选后,用PCR及RT-PCR方法测hVEGF121cDNA在基质细胞中的整合及转录;用免疫印迹及免疫组化检测VEGF121蛋白的表达;用MTT检测转基因细胞培养上清中VEGF的促人内皮细胞增殖活性.结果经酶切鉴定及基因测序证实重组逆转录病毒质粒正确,包装的病毒滴度为6×105.PCR证实基因组DNA有hVEGF121cDNA的整合;RT-PCR证实有hVEGF121mRNA的转录;Western blot及免疫组化检测有VEGF121蛋白的表达;MTT试验显示转基因细胞培养上清中VEGF121能促进人内皮细胞增殖.结论我们成功地构建了pLXSN/VEGF121重组逆转录病毒载体,该载体不仅能够有效地表达VEGF121蛋白,且表达的蛋白具有促进人内皮细胞增殖的生物学活性,这些均为进一步应用该载体研究VEGF基因治疗骨科缺血性疾患打下了基础.%Objective To construct a retroviral vector carrying human vascular endothelial growth factor (hVEGF121) cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease.Methods hVEGF121 cDNA was obtained from the plasmid pCDI/VEGF121 and cloned into retroviral plasmid pLXSN. Recombinant plasmid was transferred to the retrovirus packaging cell, PT-67, by lipofectamine mediated gene transfer. Mouse bone marrow stromal cells (MSCs) were transfected by the retrovirus. The integration of the hVEGF121 cDNA into MSC genomic DNA and expression of the VEGF gene was detected. Proliferation assays of human umbilical vein endothelial cells (HUVECs) by VEGF121 in culture medium were performed.Results Recombinant pLXSN/VEGF121 was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. hVEGF121 gene was integrated into MSC genomic DNA after transfection, and the VEGF121 protein was expressed. Proliferation assays showed VEGF121 in culture medium was a biologically active protein and had a mitogenic effect on HUVEC.Conclusions Recombinant retroviral vector carrying hVEGF121 cDNA was successfully constructed. VEGF121 protein expressed by MSCs had mitogenic effect biologically. This provides a further foundation for VEGF gene therapy for bone ischemic disease and bone tissue engineering.