增强型绿色荧光蛋白基因转染对原代培养的人成纤维细胞细胞周期的影响

目的:观察转染增强型绿色荧光蛋白(EGFP)基因后对原代培养的人瘢痕疙瘩成纤维细胞细胞周期的变化,建立原代培养的人瘢痕疙瘩成纤维细胞的示踪方法.方法:在大肠杆菌中扩增pEGFP-N1质粒,采用电转仪将pEGFP-N1质粒转入原代培养的人瘢痕疙瘩成纤维细胞,应用荧光显微镜观察其转染过程及瞬时表达情况,流式细胞仪检测其转染效率和细胞周期的变化.结果:增强型绿色荧光蛋白基因在转染24h后得到了明显表达,48h后流式细胞仪检测其表达率为32.6%,细胞周期无明显的变化,且未影响成纤维细胞的贴壁过程.结论:经pEGFP-N1质粒转染的原代培养的人瘢痕疙瘩成纤维细胞仍能在体外存活,对成纤维细胞的生长没有明显的影响.pEGFP-N1是转染原代培养的人成纤维细胞较为理想的瞬时表达载体,是研究成纤维细胞生物学行为的良好示踪剂.

Effect of EGFP gene transfection on the cell cycle distribution of primary cultured human fibroblasts

Objective:To investigate the effect of enhanced green fluorescent protein(pEGFP)gene transfection on the cell cycle distribution of primary cul- tured human fibroblasts in order to establish a tracking method of cultured human fibroblasts.Method:pEGFP-N1 was amplified in E.coli,and purified by high purity kit.Primary cultured human fibroblasts,which were initially obtained from the scars,were cultured in vitro and transferred with pEGFP-N1 by means of electroporation.Transferring process and transient expression were evaluated by fluorescence microscope.The transfer efficiency and the cell cycle distri- bution were evaluated by flow cytometry.Results:There was significant expression of EGFP at 24h after transferring.The transfection efficiency of pEGFP-N1 into primary cultured human fibroblasts reached 32.6% at 48h.It didn't affect the process of cell adherence and had no effect on the cell cycle distribution.Con- clusion:Primary cultured human fibroblasts,which were transfected with pEGFP-N1,are alive in vitro,and the transferring process doesn't affect the cell cycle distribution.These results suggested that pEGFP-NI is an ideal transient expression vector for primary cultured human fibroblasts and it might be a well tracer in studying fibroblasts.