增强型绿色荧光蛋白基因转染小鼠胚胎干细胞及其无血清神经细胞诱导  

TRANSFECTION OF MOUSE EMBRYONIC STEM CELLS WITH ENHANCED GREEN FLUORESCENT PROTEIN GENE AND ITSSERUM-FREE NEURAL DIFFERENTIATION

作　　者：徐海伟[1] 范晓棠[2] 曹娟[1] 唐军[1] 黎海蒂[1] 吴旋[1] 

机构地区：[1]第三军医大学基础医学部生理学教研室,重庆400038 [2]第三军医大学神经生物学教研室,重庆400038

出　　处：《解剖学报》2004年第5期544-547,共4页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目 ( 3 990 0 169;3 0 0 70 72 7;3 0 10 0 0 87) ;全军"十五"青年基金资助项目( 0 1Q0 99) ;重庆市 2 0 0 1年应用基础基金资助项目

摘　　要：目的　构建表达绿色荧光蛋白的小鼠胚胎干细胞 (MES)克隆 ,观察基因转染对MES细胞增殖和无血清诱导为nestin阳性神经前体细胞 (NPCs)的影响。　方法　原代分离小鼠胚胎成纤维细胞并经丝裂霉素 C处理作为饲养层细胞。质粒的转化、抽提、纯化。电穿孔法基因转染 ,MES细胞克隆的NBT/BClP染色 ,NPCs的nestin免疫组织化学染色。　结果　EGFP基因转染后可得到表达绿色荧光蛋白的MES细胞克隆 ,采用N2或ITSF无血清条件培养基可将其定向诱导为nestin阳性的并能发绿色荧光的NPCs。基因转染后的MES细胞增殖、神经前体细胞诱导率和未转染组相比均无明显下降 ,但NPCs绿色荧光的表达较诱导前明显下降。　结论　基因转染对ES细胞的增殖和神经分化无明显影响。Objective To construct the mouse embryonic stem(MES) cell line which expressed enhanced green fluorescent protein(EGFP) and to observe the influence of gene transfection on the proliferation,neural differentiation rate of MES cells. Methods Mouse embryonic fibroblast(MEF) treated with mytomycin-c was used as feeders layer.Routine methods were used in the transformation,extraction and purification of the plasmid pIRES-EGFP.The vector was transfected into MES by electroporation.The mouse MES cells were stained with NBT/BCIP,and neural precursor cells(NPCs) were stained with immuocytochemistry of nestin. Results After transfection,screening with flow cytometer,the MES was expressed stably EGFP.Cultured in the serum-free conditioned media N2 or ITSF,the MES/EGFP was differentiated into NPCs which were nestin-positive and expressed EGFP.The proliferation and the NPCs differentiation rate of the MES/EGFP did not decrease significantly compared with MES,while the EGFP expression of NPCs was decreased significantly compared with MES/EGFP.Conclusion The transfection did not influence the proliferation and neural differentiation rate of MES.

关 键 词：胚胎干细胞 神经前体细胞 绿色荧光蛋白 免疫细胞化学 小鼠 

分 类 号：Q813.11[生物学—生物工程]