增强型绿色荧光蛋白在人软骨细胞中的表达及修复重建人软骨示踪方法的研究

Expression of enhanced green fluorescent protein gene in human chondrocytes and a tracking method for reconstructing human cartilage

目的:检测增强型绿色荧光蛋白(enhancedgreenfluorescenceprotein,EGFP)基因在原代培养的人鼻中隔软骨细胞的转染效率及瞬时表达,建立原代培养的人鼻中隔软骨细胞的示踪方法,为用组织工程法修复重建人软骨寻求示踪方法提供依据。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,应用荧光显微镜和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。结果:增强型绿色荧光蛋白在基因转染24h后得到了明显表达,48h后流式细胞仪检测其转化率为35.37%,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软骨细胞仍能在体外存活,pEGFP-N1是转染原代培养的人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。

AIM:To determine the transfection efficiency and transient expression of enhanced green fluorescent protein(EGFP) gene in human nasal septal chondrocytes cultured primarily,and to build up a tracking method of the cultured human nasal septal chondrocytes so as to provide a basis for the tracking method of reconstruction and repair of tissue engineered human cartilage. METHODS:pEGFP N1 plasmid was amplified in Escherichia coli.Primary cultured human chondrocytes,which were initially obtained from the nasal septal cartilage,were cultured in vitro and transfected with pEGFP N1 by means of electroporation with Amaxa nucleofector.Transfection process and transient expression were evaluated by fluorescent microscopy and laser scanning confocal microscope(LSCM),and the transfection efficiency was evaluated by flow cytometry. RESULTS:There was obvious expression of EGFP N1 at 24 hours after transfection.The transfection efficiency of pEGFP N1 into primary cultured human chondrocytes reached to 35.37% at 48 hours.The transfection did not affect the process of cell adherence. CONCLUSION:Primary cultured human chondrocytes after transfection with pEGFP by means of electroporation can survive in vitro.pEGFP N1 is an ideal transient expression vector for primary cultured human chondrocytes,and also a good tracer in constructing tissue engineered cartilage.