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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。
OBJECTIVE: To observe the changes of the cell cycle of primary cultured human nasoseptal chondrocytes after transfection with enhanced green fluorescent protein (EGFP) gene and to establish a primary tracer of human nasoseptal chondrocytes. Methods: The pEGFP-N1 plasmid was amplified in E. coli. The pEGFP-N1 plasmid was transfected into primary cultured human nasal septum chondrocytes by Amaxa nuclear transfection instrument. The transfection process and transient expression were observed by laser scanning confocal microscopy. Flow cytometry was used to detect the transfection efficiency and cell cycle changes. RESULTS: The enhanced green fluorescent protein (EGFP) gene was expressed 24 h after transfection. The expression of enhanced green fluorescent protein (EGFP) was detected by flow cytometry after 48 h and the cell cycle was not significantly changed (35 37%). The expression of EGFP gene did not affect the attachment of chondrocytes process. CONCLUSION: Primary cultured human septal chondrocytes transfected with pEGFP-N1 plasmid can still survive in vitro and have no significant effect on the growth of chondrocytes. PEGFP-N1 is ideal for transfection of primary cultured human nasoseptal chondrocytes Of the transient expression vector, but also a good tracer of tissue-engineered cartilage formation process.