摘要
目的 将人血管内皮生长因子 (hVEGF)基因导入原代离体成人皮肤成纤维细胞 ,验证转基因细胞分泌hVEGF情况 ,探讨成人自体细胞基因修饰后移植的可行性。方法 采用酶消化法获取成纤维细胞 ,以杜氏改良培养基 (DMEM )传代培养。以脂质体转染法将pcDNA3 .1(+ ) /hVEGF12 1导入成纤维细胞 ,培养 48h后取细胞作逆转录 多聚酶链式反应 (RT PCR) ,上清行酶联免疫吸附反应 (ELISA)检测、MTT测定和Mile’s实验。结果 转基因细胞经RT PCR扩增出一条约 44 4bp条带 ,ELISA显示转基因细胞培养上清VEGF浓度约 (12 .3 2± 0 .3 9) μg/L ,MTT显示上清明显促内皮细胞增殖 ,Mile’s实验显示上清明显增加毛细血管通透性。结论 质粒 pcDNA3 .1(+ ) /hVEGF12 1成功转染成人皮肤成纤维细胞 ,转基因细胞能表达分泌有生物活性的VEGF12 1蛋白。
Objective To transfect the hVEGF121 gene plasmid into adult dermal fibroblasts and to investigate the expression and secretion of hVEGF121 gene and the biological activity of hVEGF121 protein in transfected fibroblasts.Methods The adult dermal fibroblasts were isolated,cultured in DMEM with 10% fetal bovine serum (FBS) and were identified by morphology and immunohistochemistry.The plasmid pcDNA3.1(+) hVEGF121 was transfected into fibroblasts in VEGF free medium mediated by Lipefectin2000.After being cultured for 48 h,the transcription and expression of hVEGF121 gene in transfected fibroblasts was detected by RT PCR and agarose gel electrophoresis analysis,and the hVEGF121 protein level in supernatant of transfected fibroblasts culture was determined by enzyme linked immunosorbent assay (ELISA).The biological activity of hVEGF121 protein was tested by observing the growth rate of the human umbilical vein endothelial cells (HUVEC) after being stimulated with the above supernatant,and by performing the Mile's assay in Kun ming rats.Results Adult dermal fibroblasts were successfully isolated and cultured in vitro steadily and rapidly.With Lipefectin 2000,the hVEGF121 gene in plasmid pcDNA3.1(+) hVEGF121 was successfully transfected into cultured fibroblasts in vitro.These transfected cells could secrete hVEGF121 protein to some extent,with biological activities to enhance the growth of HUVEC in vitro and improve vascular permeability.Conclusion Adult dermal fibroblasts in vitro transfected with exogenetic hVEGF121 cDNA can express and secrete active hVEGF121.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2004年第12期1539-1541,共3页
Chinese Journal of Experimental Surgery
