人VEGF165基因转染成人成肌细胞的实验研究

Human VEGF165 Gene Transfected Adult Myoblasts in vitro

目的:将人血管内皮生长因子165(hVEGF165)导入原代离体成肌细胞,观察该细胞hVEGF分泌情况,探讨成人自体转基因成肌细胞移植的可行性。方法:采用两步消化法对成人骨骼肌组织消化获取相对较纯的成肌细胞,通过差速贴壁法进行进一步的纯化。以脂质体转染法将pcDNA3.1-hVEGF165导入成肌细胞,通过RT-PCR、ELISA和Western-blot进行hVEGF165定量检测,MTT测定和Mile's实验检测VEGF165的生物学活性。结果:转基因细胞经RT-PCR扩增出一条VEGF的特异性泳带,ELISA显示转基因细胞培养上清VEGF浓度分别达到18.92±1.77 ng/mL、19.04±2.15 ng/mL,Western blot检测转基因成肌细胞上清均检测到VEGF蛋白特异性的杂交带,MTT显示转基因细胞上清明显促内皮细胞增殖,Mile's实验显示转基因细胞上清明显增加毛细血管通透性。结论:质粒pcDNA3.1-hVEGF165能成功转入成人成肌细胞,转基因细胞能分泌有生物活性的VEGF165蛋白。

Objective：To transfect the hVEGF165 gene plasmid into adult skeletal myoblasts and to investigate the expression and secretion of hVEGF165 gene and the biological activity of hVEGF165 protein in transfected myoblasts. Methods ： The myoblasts were isolated from human skeletal muscle with two steps combined dissociation, then the harvested myoblasts were purified by the method Of different attachment. The ptasmid pcDNA 3.1-VEGF165 was transfected into myoblasts in free medium mediated by Lipefectin 2000. After being cultured for 48 h, the transcription and expression of hVEGF165 gene in transfected myoblasts were detected by RT-PCR and agarose gel electrophoresis analysis, and the hVEGF165 protein level in supernatant of transfected myoblasts culture was determined by western blot and enzyme linked immunosorbent assay （ELISA） . The biological activity of hVEGF165 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 Kunming rats. Results： Adult myoblasts were successfully isolated and cultured in vitro steadily and rapidly. With Lipefectin 2000, the hVEGF165 gene in plasmid pcDNA 3.1-hVEGF165 was successfully transfected into cultured myoblasts in vitro. These transfected cells could secrete hVEGF165 protein to certain extent, with biological activities to enhance the growth of HUVEC in vitro and improve vascular permeability. Conclusion： Adult myoblasts in vitro transfected with exogenetic hVEGF165 cDNA can express and secrete active hVEGF165.