绿色荧光蛋白标记神经干细胞的体外研究

目的　构建携带绿色荧光蛋白 (GFP)基因的逆转录病毒载体pLNCX2 GFP ,用GFP对神经干细胞进行标记示踪。方法　应用基因克隆的方法 ,制备 pLNCX2 GFP ,借助阳离子脂质体转染包装细胞PA3 17,G418筛选阳性克隆 ,获取病毒上清 ;从胚胎大鼠脑中解剖分离和培养神经干细胞 ,用病毒上清感染大鼠胚胎神经干细胞。结果　经酶切电泳和DNA测序表明成功构建了重组GFP逆转录病毒 ,pLNCX2 GFP转染包装细胞后可以产生GFP逆转录病毒 ,病毒感染大鼠胚胎神经干细胞可以长期表达绿色荧光。结论　逆转录病毒能够快速、稳定、长期地将GFP基因转入神经干细胞 ,这种标记方法非常有利于神经干细胞移植后结构和功能的研究

Construction of green fluorescent protein retroviral vector and label of rat ebryonic neural stem cell

Objective To construct retroviral vector pLNCX2-GFP carrying green fluorescent protein (GFP) and to label rat fatal neural stem cells (NSCs) for further structure analysis and physiological recordings following transplantation.Methods Recombinant retroviral vector pLNCX2-GFP was constructed by directionally cloning GFP cDNA from pEGFP-N1 into the MCS of the retroviral vector pLNCX2.pLNCX2-GFP plasmid was transfected into packaging cell line PA317 by Lipofectamin.G418-resistant clones were selected in 4 weeks and viral stocks were gathered.Retroviral titer was tested by counting the number of NIH3T3 cells that expressed GFP observed under fluorescence microscopy.Fetal neural stem cells (NSCs) were isolated from the brain of 14-16 days embryonic SD rats,and cultured in serum-free medium with EGF,bFGF and supplements B27 after digestion.The recombinant viruses were used to infect NSCs.Results The recombination retroviral vector pLNCX2-GFP carrying selective marker GFP was constructed and was confirmed by agarose gel electrophoresis and DNA sequencing.GFP expression in packaging cell line PA317 transferred by pLNCX2-GFP by Lipofectamin was detected by fluorescence microscopy.Recombinant viruses were produced following transferred into packaging cell.Conclusion Retroviral vector can transferred the GFP cDNA into the fetal NSCs quickly and stably.This way of labeling is favorable for the structure analysis and physiological recordings following cell therapy in treatments of neurological disorders.