摘要
目的建立增强型绿色荧光蛋白(EGFP)基因修饰大鼠胚胎中脑神经干细胞。方法以质粒pEGFPN1转染培养第三代的大鼠胚胎中脑神经干细胞(mNSCs),经G418筛选后,分别进行nestin免疫细胞化学鉴定和诱导分化后β-Ⅲ-tubulin、GFAP、CNPase免疫细胞化学鉴定。结果 EGFP在基因转染12 h后开始表达,24 h后明显增加,48 h达到高峰,经G418筛选1月后有阳性克隆形成,EGFP基因修饰不影响大鼠胚胎mNSCs的增殖与分化。结论成功建立EGFP基因修饰大鼠胚胎mNSCs,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 enhanced green fluorescent protein （EGFP） gene modified midbrain-derived neural stem ceils （mNSCs）. Methods The E14 rat embryonic mNSCs were isolated and cultured. The ceils of the third passage were transfected with plasmid pEGFPN1 using FuGENE HD transfection reagent. The expressions of EGFP were observed under fluorescence microscope. 48 hours after transfection, the transfected cells were screened with medium containing G418. The positive clones were selected, proliferated and then identified with immunocytochemistery for nestin. At the same time, the cells were inducted to differentiation. The distinctive marker for neuron （ρ-Ⅲ-tubulin）, dopaminergic neuron （ TH）, astrocyte （GFAP） and oligodendrocyte （CNPase） were employed to detect the phenol type of differentiated cells. Results The expression of EGFP was initially found 12 hours after transfection, increased remarkable 24 hours after transfection and reached a summit at 48 hours. One month after screened with medium containing G418, the positive clones were formed. The immunocytochemistery showed the cells were nestin positive, after differentiation the cells expressed ρ-Ⅲ-tubulin, TH, GFAP and CNPase. Conclusions The EGFP gene modified embryonic mNSCs in rats were successfully established, which will provide the foundation for the further research about cell therapy of Parkinson＇ s disease.
出处
《临床神经外科杂志》
CAS
2010年第3期116-118,共3页
Journal of Clinical Neurosurgery
基金
云南省教育厅科学研究基金(09Y0153)
昆明医学院第一附属医院博士启动基金
关键词
增强型绿色荧光蛋白基因
中脑神经干细胞
基因转染
enhanced green fluorescent protein gene
midbrain-derived neural stem cells
transfection
