EGFP基因修饰大鼠胚胎中脑神经干细胞的建立.pdfVIP

116 JClinNeurosurg，September2010，Vo1．7，No．3 doi：10．3969／j．issn．1672-7770．2010．03．002 · 论 著 · EGFP基因修饰大鼠胚胎 中脑神经 干细胞的建立 邓兴力，杨智勇，宋晓斌，蔡山，李智高，王永刚，刘如恩，冯忠堂 【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p—Ill-tubulin、GFAP、CNPase免疫细胞化学鉴定。结果 EGFP在基因转染 12h后开 始表达，24h后明显增加，48h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。 【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 【中图分类号】 R742．5 【文献标识码】 A 【文章编号】 1672．7770(2010)03-0116-03 EstablishmentofEGFP genemodifled embryonicmidbrain,derived neuralstem cellsin ratsDENG 一li，YANGZhiy·0 ，SONGXiao-bin，eta1．DepartmentofNeurosurgery，theFimt AffiliatedHospitalofKunmingMedicalCollege，Kunming650032，China Abstract：0bjective Toestablishenhancedgreenfluorescentprotein(EGFP)genemodified midbrain—derivedneuralstem cells(mNSCs)．Methods TheEl4ratembryonicmNSCswere isolatedandcultured．ThecellsofthethirdpassageweretransfectedwithplasmidpEGFPN1using FuGENE HD transfection reagent． The expressionsofEGFP were observed under fluorescence microscope．48hoursaftertransfection，thetransfectedcellswerescreenedwithmedium containing G418． The positive clones were selected． proliferated and then identified with immunocytochemisteryfornestin．Atthesametime．the cellswere inductedto differentiation．The distinctivemarkerofrneuron (31-III—tubulin)，dopaminergicneuron (TH)，astrocyte(GFAP)and oligodendrocyte(CNPase)wereemployedtodetectthephenoltypeofdifferentiatedcells．Results Theexpression ofEGFP wasinitially ofund 12 hoursaftertransfection，increased remarkable24 hoursaftertransfectionandreachedasummitat48hours．Onemonthafterscreenedwithmedium containingG418，thepositivecloneswereformed．Theimmunocytochemisteryshowedthecellswere nestin positive，after differentiation the cellsexpressed 3-II