Nestin-GFP小鼠胚胎干细胞的建系及体外神经分化  

Establishment and Neural Differentiation of Nestin-GFP Mouse Embryonic Stem Cells

作　　者：王紫菲[1] 赖文玉[2] 柯琼[1] 李付贵[1] 王涛[1] 张秀明[1] 李伟强[1] 项鹏[1] 

机构地区：[1]中山大学干细胞与组织工程研究中心,广东广州510080 [2]中山大学附属第二医院儿科,广东广州510120

出　　处：《中山大学学报（医学科学版）》2011年第2期155-163,共9页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省自然科学基金博士启动(9451008901002230)

摘　　要：【目的】建立Nestin-GFP转基因小鼠的胚胎干细胞(mESC)系,并观察其示踪ES细胞系的神经分化过程。【方法】将E3.5的Nestin-GFP小鼠囊胚接种于小鼠胚胎成纤维细胞上(MEF经γ射线照射失去增殖活性)。4～6d后将自囊胚长出的内细胞团(ICM)继续培养并传代扩增,检测所得细胞Oct-4、SSEA-1和AKP等胚胎干细胞特异性标志物的表达及在体内外向三胚层分化的能力;之后,诱导细胞向神经元样细胞及神经胶质样细胞分化,利用免疫荧光检测其标志性抗原。【结果】免疫组化可见Nestin-GFP小鼠胚胎干细胞表达未分化细胞特异性标志Oct-4、SSEA-1,AKP染色呈强阳性,并具有向体内外三胚层分化潜能;在体外诱导其向神经分化,第6d可见到明显的绿色荧光,并与免疫组化Nestin表达部位基本吻合。第12d及第16d分别可见神经元及神经胶质细胞特异性标志物表达,而此时Nestin的表达较前明显下降。【结论】成功建立了Nestin-GFP小鼠胚胎干细胞系,该细胞具有自我更新的特性及多向分化潜能,并可用于在体外更直观的监测神经分化的阶段性变化并由此进行调控。[Objective] To establish a mouse embryonic stem cell line which contains the green fluorescent protein（GFP） reporter under the control of Nestin promoter,and identify the stages of differentiation of neural cells by direct observation of GFP expression.[Methods]The E3.5 blastocysts from Nestin-GFP female mice were placed onto MEF feeder layer（mouse embryonic fibroblasts）（inactivated by γ-ray） and cultured for 4-6 days.Then the inner cell mass（ICM） was isolated and passaged.Expression of Oct-4,SSEA-1,AKP,and differentiation into three germ layers in vivo and in vitro were detected for characterization of Nestin-GFP mouse embryonic stem cells（mESC）.And the neural differentiation of Nestin-GFP mESC into neurons and glial cells was also identified by the immunocytochemical assays.[Results]Immunofluorescence staining demonstrated that these cells expressed the undifferentiated marker Oct-4 and SSEA-1,as well as the AKP.The results also proved that Nestin-GFP mESC still retained the pluripotency of differentiation into three germ layers both in vivo and in vitro.The GFP expression was evident as early as 6 days during neural differentiation in vitro,which correlated closely with endogenous Nestin expression.After that,GFP expression was down regulated and Nestin positive cells were replaced by the βⅢ-tubulin＋ and GFAP＋ cells at day 12 and day 16.[Conclusion]The Nestin-GFP mESC not only maintained the distinct characteristics of ESC,but also exerted beneficial influence on the visualized observation and regulation of the dynamic phase changes during the neural differentiation.

关 键 词：NESTIN 胚胎干细胞 绿色荧光蛋白 神经分化 

分 类 号：Q254[生物学—细胞生物学]