脂质体介导血管内皮生长因子基因在内皮细胞的稳定表达

The Stable Expression of Vascular Endothelial Growth Factor Gene Mediated by Lipofect AMINE in Human Umbilical Vein Endothelial Cell Line Cells

建立稳定表达血管内皮生长因子的人脐静脉内皮细胞系 ,为其在组织化工程血管的应用奠定基础。将真核表达载体PCD2 VEGF1 2 1 用阳离子脂质体介导 ,转染人脐静脉内皮细胞系细胞 ,G4 18筛选 ,获得G4 18抗性单克隆细胞 ,扩增后分别用逆转录聚合酶链反应、免疫组织化学及血管通透性实验检测血管内皮生长因子的转录、蛋白质的表达及其生物学活性。结果发现 ,逆转录聚合酶链反应检测出了转录血管内皮生长因子的稳定转染细胞克隆 ,该单克隆细胞的免疫组织化学检测血管内皮生长因子蛋白质表达呈阳性 ,血管通透性实验和细胞生长实验发现其表达产物具有生物学活性 ,而作为对照的转空白质粒细胞和未转染细胞上述实验结果皆为阴性。结果表明 。

Aim To establish a human umbilical vein endothelial cell (hUVEC) line monoclonal cells with the stable expression of vascular endothelial growth factor (VEGF) gene. Methods The eukaryotic expression vector PCD 2 VEGF 121 were transfected into hUVEC line cells mediated by lipofect AMINE. The positive clones were obtained by the screen of G 418 . The transcription and expression of VEGF gene were investigated by RT PCR and immunocytochemistry respectively. The experiment of Miles was applied for the assay of the biological activity of the protein of the VEGF produced by the hUVEC line cells which transfected PCD 2 VEGF 121 . The growth curve was made for comparison with that of non transfected hUVEC line cells. Results A positive clone cells from which transcripted the MRNA of VEGF 121 gene was obtained by RT PCR; The positive results of the immunocytochemistry were found and the highly biological activity of VEGF in the media was detected each only in the positive clone cells. The doubling time of the positive colon cells was shorter than that of the non transfected hUVEC line cells calculated from the growth curve. Conclusion The hUVEC line monoclonal cells with the stable expression of VEGF gene has been established successfully.