脂质体介导的VEGF_(165)基因转染对内皮细胞生长的作用

构建血管内皮细胞生长因子基因VEGF165真核表达载体pcDNA3 VEGF165,以阳离子脂质体介导的基因转染技术 ,将基因转入原代培养的人脐静脉内皮细胞中。结果表明 ,基因转染 1h后细胞内就有VEGFDNA存在 ,VEGFmRNA水平显著上升 ;基因转染 2d后培养液上清VEGF蛋白表达显著上升 (135 5 12± 6 2 34)pg/ml和 (19 2 7± 2 96 )pg/ml,P <0 0 1。转染VEGF165基因 2d的内皮细胞再经程序降温冷冻保存复苏后 ,其存活率显著高于对照组 (pcDNA3 组 ) (90 13%± 2 84 %和81 5 2 %± 2 15 % ,P <0 0 5 ) ,凋亡率显著低于对照组 (7 15 %± 0 4 2 %和 17 6 1%± 1 5 6 % ,P <0 0 5 )。MTT法显示转染VEGF165基因能促进内皮细胞VEGF蛋白的表达 ,促进细胞增殖 ,抑制细胞凋亡。在治疗心脏及下肢动脉缺血性疾病中 ,VEGF165基因治疗可能具有重要的意义。

Effect of Transfection with Liposome-Mediated Vascular Endothelial Growth Factor Gene VEGF165on the Growth of Endothelial Cells

To construct the eukaryotic expression vector pcDNA 3-VEGF 165 with cationic liposome mediated gene transfection technique and transfect into the primarily cultured human umbilical vein endothelial cells (EC),it was found that the intracellular existence of the VEGF DNA could be detected one hour after gene transfection with prominent elevation in the level of VEGF mRNA as determined by PCR. Two days after transfection,the expression of the VEGF proteins in the supernatant of culture fluid significantly increased (1 355.12±62.3 vs 19.27±2.96)pg/ml. The apoptosis of EC was significantly decreased compared with pcDNA 3 transfection (7.15%±0.42% vs 17.61%±1.56%),while the survival rate was notably increased (90.13%±2.84% vs 81.52%±2.15%) as determined by flow cytometry analysis after programmed cryopreservation and thawing. As revealed in the MTT the transfected VEGF 165 gene could improve the expression of VEGF proteins in EC,accelerate cell proliferation and inhibit the development of apoptosis of EC. It is hopeful that the VEGF 165 gene therapy may be used for the treatment of the ischemic disorders of heart and lower extremities.