人血管内皮生长因子基因的克隆、表达及生物活性分析

目的 克隆人血管内皮细胞生长因子165(VEGF165)基因，构建真核表达载体，观察其对脐静脉内皮细胞的增殖作用和血管新生的影响。方法 利用RT-PCR方法，从人扁桃体组织中扩增人VEGF165 cDNA完整编码区，并构建成pcDNA3．1( )／VEGF165(简称pcDNA／V)重组体；应用脂质体介导的基因转移技术将构建的真核表达载体pcDNA／V体外转染至人脐静脉内皮细胞(HUVEC)，MTT法检测其对内皮细胞增殖的影响。建立家兔下肢缺血模型，注射重组质粒pcDNA／V，pcDNA3．1( )空质粒作对照，选取不同时间点，行血管造影。结果 构建的真核表达载体pcDNA／V的酶切电泳分析和测序表明结果正确。pcDNA／V转染HUVEC能明显促进内皮细胞的分裂增殖。血管造影显示，术后基因治疗组远端动脉充盈早于对照组，新生血管数目也明显多于同时期对照组。结论 成功克隆了人VEGF165基因，构建了其真核表达载体。体内外生物学活性研究证实，重组质粒的表达产物具有刺激HUVEC增殖和促进缺血肢体侧枝循环建立的功能。

CLONGING,EXPRESSION AND BIOLOGICAL ACTIVITY ANALYSIS OF HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR

Objective To construct eukaryotic expression vector containing vascular endothelial growth factor(VEGF) cDNA gene,which included full coding region,and study its biological activity. Methods Human vascular endothelial growth factor 165(VEGF 165 )cDNA was amplified by RT-PCR from human tonsil tissue,the VEGF 165 cDNA was sequenced and inserted into eukaryotic expression vector pcDNA*!3.1(+).The construct pcDNA*!3.1(+)/VEGF 165 (pcDNA/V)was transferred into HUVEC cells mediated by liposome in vitro.The proliferation level of VEGF 165 was detected by MTT.The hindlimb ischemia model was developed by ligation of the distal external iliac artery and excision of every branches of femoral arteries.The construct pcDNA/V was transferred by injection into the ischemia muscle,and its angiogenesis effect was observed by angiography. Results Identifications of the pcDNA/V were confirmed by digestion of endonuclease and sequence analysis.After pcDNA/V was transfered into HUVEC cells 72*!h,MTT results showed that it had activity to stimulate HUVEC proliferation.Angiography demonstrated that in the experimental animal group,the filling of collateral circulation and reconstitution of the artery was higher than that in the control group at the same time.Conclusions VEGF 165 cDNA full coding region was successfully cloned,then pcDNA*!3.1(+)eukaryotic expression vector was constructed.The construct pcDNA/V showed good biological activity to stimulate HUVEC proliferation and to promote formation of collateral circulation in a persistent hindlimb ischemia model.