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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 medium-density neural stem cells modified with superparamagnetic iron oxide (SPIO) and enhanced green fluorescent protein (EGFP) double-labeled nerve growth factor (NGF) gene. METHODS: The third generation of rat embryonic mesencephalic neural stem cells were co-transfected with plasmids pcDNA3-hNGF and pEGFPN1 and labeled with SPIO. The expression of EGFP was detected by fluorescence microscope. Immunocytochemistry and Western Blot were used to identify the expression of NGF. Prussian blue staining and transmission electron microscopy were used to identify the expression of EGFP. Results: The expression of EGFP began 12h after transfection. Immunocytochemistry and Western Blot showed that the cells could express NGF correctly. The Prussian blue staining showed that the cell labeling rate was 100%. Transmission electron microscopy showed that the SPIO particles were located in the swallow vesicles and cytoplasm . Conclusion: SPIO and EG-FP double-labeled NGF gene modified mesencephalic neural stem cells were established, which laid the foundation for the further study of cell transplantation in the treatment of Parkinson’s disease.