超顺磁氧化铁和EGFP双标NGF基因修饰中脑神经干细胞的建立

Establishment of superparamagnetic iron oxide and enhanced green fluorescence protein double labeled nerve growth factor gene modified midbrain derived neural stem cells

目的:建立超顺磁氧化铁(SPIO)、增强型绿色荧光蛋白(EGFP)双标神经生长因子(NGF)基因修饰中脑神经干细胞。方法:以质粒pcDNA3-hNGF、pEGFPN1共转染第三代大鼠胚胎中脑神经干细胞并用SPIO标记。荧光显微镜检测EGFP的表达;免疫细胞化学、Western Blot鉴定NGF的表达;普鲁士蓝染色、透射电镜鉴定SPIO标记。结果:EGFP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NGF;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:建立了SPIO、EG-FP双标记NGF基因修饰中脑神经干细胞,为进一步开展细胞移植治疗帕金森病的研究奠定基础。

Objective： To establish superparamagnetic iron oxide （SPIO） and enhanced green fluorescence protein （EGFP） double labeled nerve growth factor （NGF） gene modified midbrain neural stem cells. Methods： The rat embryonic midbrain derived neural stem cells of the third passage were co-transfected with plasmid pcDNA3-hNGF and pEGF- PN1 and labeled with SPIO. The expression of EGFP was observed with fluorescence microscope and the expression of NGF was analyzed by immunocytochemistry as well as Western Blot. Prussian blue staining and transmission electron mi- croscopy was used to identify the SPIO particles in cells. Results： The expression of EGFP was initially found 12 hours after transfection. The immunocytochemistry and Western Blot showed the NGF was expressed successfully in cells. Prussian blue staining showed the percentage of labeled cells was 100%. Transmission electron microscopy showed vacuolar structures of different sizes under the cytoplasma within and outside of which there were highly density particles. Conclusion： The SPIO and EGFP double labeled NGF 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.