VEGF shRNA表达质粒的构建及对血管内皮细胞VEGF表达的抑制

目的：构建大鼠血管内皮生长因子（VEGF）短发夹RNA（shor thairpin RNA,shRNA）真核表达质粒,观察其对大鼠主动脉血管内皮细胞（ECs）中VEGF表达的影响.方法：采用DNA重组技术根据大鼠VEGF mRNA序列设计并合成3条shRNA寡核苷酸片段,构建表达质粒并通过脂质体介导转染ECs,采用RT-PCR,Western Blot方法分别检测VEGF mR-NA及蛋白质的表达.结果：经酶切和测序证实VEGF shRNA重组质粒构建成功.3条VEGF shRNA质粒均可下调VEGF mRNA和蛋白的表达,其中VEGF shRNA3质粒的抑制作用较强,转染72h时VEGF mRNA和蛋白的抑制率可分别达到71.91%和67.86%.结论：成功构建VEGF靶向RNA干扰重组体,并能有效抑制ECs中VEGF mRNA和蛋白的表达,为利用RNA干扰技术从转录后水平抑制角膜新生血管的研究提供依据.

Construction of VEGF small hairpin RNA expression plasmid and inhibition of VEGF expression in rat vascular endothelial cells

AIM:To construct short hairpin RNA(shRNA) eukaryotic expression plasmid of rat vascular endothelial growth factor(VEGF) and to explore its effects on VEGF expression in rat vascular endothelial cells(ECs).METHODS:Three different shRNA oligonucleotides targeting the coding sequence of rat VEGF mRNA were designed and cloned into pSilencer2.0 U6 plasmid,respectively.The recombinant plasmids were then transfected into ECs via Lipofectamine 2000.Expression of VEGF was detected by RT-PCR and Western blot.RESULTS:...