【摘要】 目的 :观察转染增强型绿色荧光蛋白 (EGFP)基因后原代培养的人鼻中隔软骨细胞的细胞周期的变化 ,建立原代培养的人鼻中隔软骨细胞的示踪方法。方法 :在大肠杆菌中扩增pEGFP -N1质粒 ,通过Amaxa细胞核转染仪将pEGFP -N1质粒转入原代培养的人鼻中隔软骨细胞 ,应用激光共聚焦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 2 4h后得到了明显表达 ,4 8h后流式细胞仪检测其表达率为 35 37% ,细胞周期没有明显的改变 ,且未影响软骨细胞的贴壁过程。结论 :经pEGFP -N1质粒转染的原代培养的人鼻中隔软骨细胞仍能在体外存活 ,对软骨细胞的生长没有明显的影响 ,pEGFP -N1是转染原代培养的人鼻中隔软骨细胞较为理想的瞬时表达载体 ,也是组织工程化软骨形成过程的良好示踪剂。更多还原

【Abstract】 AIM: To investigate the effect of enhanced green fluorescence protein (EGFP) gene transfection on the cell cycle distribution of primary cultured human chondrocytes in order to establish a tracking method of cultured human nasoseptal chondrocytes. METHODS: pEGFP-N1 plasmid was amplified in E.coli, and purified by high purity kit. Primary cultured human chondrocytes,which were initially obtained from the nasoseptal cartilage, were cultured in vitro and transferred with pEGFP-N1 by means of electroporation with Amaxa nucleofector device. Transfering process and transient expression were evaluated by laser scanning confocal microscope (LSCM), the transfer efficiency and the cell cycle distribution were evaluated by flow cytometry. RESULTS: There was significant expression of EGFP at 24 h after transferring. The transfection efficiency of pEGFP-N1 into primary cultured human chondrocytes reached 35 37% at 48 h. It didn’t affect the process of cell adherance and had no effect on the cell cycle distribution. CONCLUSION: Primary cultured human chondrocytes, which were transfected with pEGFP, are alive in vitro, and the transferring process doesn’t affect the cell cycle distribution. These results suggest that pEGFP-N1 is an ideal transient expression vector for primary cultured human chondrocytes and it might be a well tracer in construction tissue engineered cartilage.更多还原