人VEGF165基因真核表达质粒的构建及其对血管内皮细胞增殖的影响

目的 :构建人VEGF16 5基因的真核表达质粒pBudCE4 .1/VEGF16 5 ,观察其在血管内皮细胞 (VEC)中的表达和表达产物对VEC增殖的影响。方法 :用RT PCR法从引产胎儿心脏组织中克隆VEGF16 5基因 ,并将其克隆至真核表达质粒pBud CE4 .1中 ,对重组真核表达质粒pBudCE4 .1/VEGF16 5进行酶切鉴定和测序。以脂质体转染法将pBudCE4 .1/VEGF16 5导入VEC中 ,用Northernblot和免疫细胞化学染色法 ,分别从mR NA水平和蛋白质水平检测它们在转染的VEC中的表达 ,并检测表达产物对VEC增殖的影响。结果 :人VEGF16 5基因的RT PCR产物为 5 76bp。测序结果显示 ,扩增的VEGF16 5基因的序列与基因文库中登录的序列完全一致。经HindⅢ和BamHI酶切鉴定证实 ,VEGF16 5基因已成功地克隆至真核表达质粒pBudCE4 .1中。以其转染血管内皮细胞 (VEC)后 ,经Northernblot杂交和免疫细胞化学染色法检测均证实VEGF16 5基因表达。表达产物对VEC增殖有明显的促进作用。结论 :所构建的pBudCE4 .1/VEGF16 5真核表达质粒可在VEC中表达 ,表达产物可明显促进VEC增殖 ,为通过VEGF16 5基因转染防治移植器官内血管的狭窄奠定了基础

Effect of human VEGF165 in vitro on proliferation of vascular endothelial cells

AIM: To observe the effect of human VEGF165 on the proliferation of vascular endothelial cells(VECs) in vitro. METHODS: VEGF165 gene was amplified by RT-PCR and cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme(Hind III and BamH I) digestion analysis and confirmed by DNA sequencing. The pBudCE4.1/VEGF165 was transfected into VECs through lipofection transfection. Expression of VEGF165 was detected by Northern blot and immunocytochemical staining. The effect of expressed VEGF165 on VEC proliferation was detected by flow cytometry. RESULTS: Sequencing analysis revealed that the sequence of cloned VEGF165 gene was identical with that in GenBank (accession No.X62568). Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/tVEGF165 was constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transfected VECs was demonstrated by Northern blot and immunocytochemical staining, respectively. The expressed VEGF165 notably stimulated the proliferation of VECs. CONCLUSION: pBudCE4.1/VEGF165 is successfully constructed and expressed in VECs. Expressed VEGF165 can stimulate the VEC proliferation. The present study lays the foundation for using VEGF165 gene transfection to prevent and treat vascular stenosis in transplanted organ.