摘要
目的研究腺相关病毒载体介导人血管内皮生长因子基因(AAV-VEGF121)的体外促血管内皮细胞生长作用。方法 RT-PCR法获取人VEGF121基因,克隆入pAAV-MCS载体中,构建成pAAV-VEGF121(处理组),以空白载体克隆入pAAV-MCS病毒(对照组);两组分别在AAV-293细胞中包装成pAAV-VEGF121病毒颗粒和空白病毒颗粒。相同条件下,两组分别感染人脐静脉内皮细胞(HUVEC),半定量RT-PCR检测细胞内VEGF121基因mRNA的表达;CCK-8法测取OD450nm值,绘制细胞增殖曲线;电子显微镜观察细胞超微结构的改变。结果成功构建AAV-VEGF121;半定量RT-PCR显示处理组细胞内VEGF121的mRNA表达水平高于对照组(P<0.01);细胞增殖曲线显示处理组细胞增殖能力明显高于对照组;透射电镜显示处理组细胞内与蛋白质合成有关的细胞器增生明显活跃。结论 AAV-VEGF121基因对血管内皮细胞的生成具有促进作用。
Objective To study the angiogenesis effect of vascular endothelial growth factor gene-121（VEGF121）mediated by recombinant adeno-associated viral vector（rAAV） on vascular endothelial cells in vitro.Methods The human VEGF gene-121 was obtained by RT-PCR and directly cloned into the pAAV-MCS to form plasmid pAAV-VEGF121 which was packaged into pAAV-VEGF121 viral particles in AAV-293 cells（treatment group）.pAAV-MCS virus was cloned with a blank vector（control group）.Both were used to infect human umbilical vein endothelial cells（HUVECs）.VEGF121 mRNA expression was detected by semi-quantitative RT-PCR.Cell proliferation assay was performed using cell counting Kit-8（CCK-8） and electron-microscope was applied to observe ultrastructure changes in the cells.Results The recombinant AAV expressing human VEGF121 gene was efficiently constructed.Semi-quantitative RT-PCR and cell proliferating assay revealed higher VEGF121 mRNA level（P0.01） and stronger proliferating ability in the cells of treatment group. Electron-microscope result also showed a greater proliferating activity of organells concerning protein synthesis in the cells of treatment group.Conclusion This study has verified the fact that AAV vector-mediated VEGF121 gene has angiogenesis effect on vascular endothelial cells in vitro.
出处
《江苏医药》
CAS
CSCD
北大核心
2010年第18期2204-2208,共5页
Jiangsu Medical Journal
关键词
血管内皮生长因子
腺相关病毒载体
Vascular endothelium growth factor
Adeno-associated viral vector
