摘要
目的 建立超顺磁氧化铁（superparamagnetic iron oxide，SPIO）、绿色荧光蛋白（green fluorescent protein，GFP）双标神经营养素3（neurotrophin 3，NT-3）基因修饰中脑神经干细胞。方法 以质粒pcDNA3-NT3、pGFPN1共转染第三代E14大鼠胚胎中脑神经干细胞并用SPIO标记。荧光显微镜检测GFP的表达；免疫细胞化学、western blot鉴定NT-3的表达；普鲁士蓝染色、透射电镜鉴定SPIO标记。结果 GFP在基因转染12h后开始表达；免疫细胞化学、western blot表明细胞能正确表达NT-3；普鲁士蓝染色显示细胞标记率达100％，透射电镜显示SPIO颗粒位于吞饮小泡和胞浆内。结论建立SPIO、GFP双标NT-3基因修饰中脑神经干细胞，为进一步开展帕金森病的细胞移植治疗研究奠定基础。
Objective To establish superparamagnetie iron oxide（SPIO） and enhanced green fluorescence protein（EGFP） double labeled neurotrophin 3（NT-3） gene modified neural stem cells. Methods The El4 rat embryonic midbrain derived neural stem cells were isolated and cultured. The cells of the third passage were co-transfected with plasmid pcDNA3-NT3 and pEGFPN1 using FuGENE HD transfection reagent and screened with medium containing G418. The positive clones were selected, proliferated and then labeled with SPIO mediated by FuGENE HD transfection reagent. The expression of EGFP was observed with fluorescence microscope and the expression of NT-3 was analyzed by immunocytochemistry as well as western blot. Prussian blue stain and transmission electron microscope was used to identify the SPIO particles in cells. Results The expression of EGFP was initially observed 12 hours after transfection and increased remarkably 24 hours after transfection and reached to the peak at 48 hours. The positive clones were estanlished one month after screening with medium containing G418, Immunoeytochemistry and western blot showed that the NT-3 was expressed successfully in the cells. Prussian blue staining showed numerous blue stained particles in the cytoplasma of the labeled cells. Transmission electron microscopy showed that the SPIO particles were located in the vacuolar structures and the cytoplasma. Conelnsion The SPIO and EGFP double labeled NT-3 gene modified midbrain derived neural stem cells were successfully established, which will provide the foundation for the further research about cell therapy of Parkinson disease
出处
《国际生物医学工程杂志》
CAS
2008年第1期15-18,F0003,共5页
International Journal of Biomedical Engineering
关键词
超顺磁氧化铁
绿色荧光蛋白基因
神经营养素3基因
中脑神经干细胞
Superparamagnetic iron oxide
Green fluorescent protein gene
Neurotrophin 3 gene
Midbrain derived neural stem cells
