VEGF重组质粒pcDNA/V的构建及其在大鼠急性心肌缺血模型中的表达

构建人血管内皮细胞生长因子(VEGF165)真核表达载体,并研究其在细胞水平和大鼠急性心肌梗死动物模型中的表达。利用RT-PCR方法,从人扁桃体组织中扩增人VEGF165基因,构建真核表达载体pcDNA/V。应用脂质体介导的基因转移技术,将pcDNA/V转染至人胚肾细胞(293细胞)中,经G418筛选获得稳定表达的重组质粒细胞克隆。ELISA、Westernblot检测证实重组质粒pcDNA/V能在293细胞中高效表达外源VEGF基因,鸡胚绒毛尿囊膜血管生成实验证实表达产物具有促血管生成的活性。进一步的体内表达研究,建立大鼠急性心肌梗死模型,将重组质粒pcDNA/V、空质粒pcDNA3.1( )分三点注射于梗死交界处心肌内,四周后取材。经免疫组化染色检测,pcDNA/V组在梗死交界区有VEGF阳性表达;电镜观察显示,pcDNA/V组在梗死交界处心肌细胞间有大量毛细血管内皮细胞增生。实验结果表明成功克隆了人VEGF165基因,构建了其真核表达载体。体内、外表达研究证实重组质粒的表达产物具有促血管生成的生物学活性,为VEGF基因治疗缺血性心肌病的研究提供实验基础。

Construction of VEGF Recombinant Plasmid pcDNA/V and its Expression in Model Rats with Acute Myocardial Ischemia

The cDNA encoding human Vascular Endothelial Growth Factor 165 (VEGF165) was amplified using RT-PCR from human tonsil tissue and cloned into eukaryotic expression vector pcDNA3.1 (+). The recombinant plasmid pcDNA/V was transferred into 293 cells mediated by liposome and the cells stably expressing VEGF were selected under the pressure of G418. ELISA and Western blotting demonstrated that the eukaryotic expression vector pcDNA/V was successfully constructed and its corresponding protein could be expressed efficiently in vitro. Chick Charioallantoic Membrane (CAM) bioassay showed that recombinant protein has biological activity of hVEGF. Model rats with acute myocardial ischemia were used to further study the expression of VEGFin vivo. The model rats were divided randomly into three groups: control group, pcDNA3.1 (+) group and pcDNA/V group. 50microL naked plasmid DNA or saline was intramyocardially injected at three sites into the border zone of infarction. The hearts of rats were excised and fixed histologically, then the infarction sizes were studied by immunohistochemical staining and electron microscope after four weeks. Immunohistochemical staining for VEGF appeared to be negative in control and pcDNA3.1 (+) groups. In pcDNA/V group, myocardial cells in infarction border zone showed positive staining for VEGF in cytoplasm. Ultrastructural anaylsis showed that there were visible hyperplasia of vascular endothilium in pcDNA/V group. The control and pcDNA3.1 (+) groups showed less capillary hyperplasia. In this study, VEGF165 gene was successfully cloned and its protein expressed in vitro and in vivo was of bioactivity, which provides a basis for the further study of biological functions of human VEGF.