昆明小鼠胚胎干细胞定向分化为神经干细胞的实验研究

Differentiation of Kunming mouse embryonic stem cells into neuronal stem cells in vitro

目的探讨体外分离培养、诱导胚胎干细胞(ESC)分化为神经干细胞(NSC)的方法。方法自孕3.5d的昆明小鼠获得附植前的早期胚胎,在小鼠胚胎成纤维细胞(MEF)饲养层上培养获得ESC克隆。进行Oct-4免疫细胞化学鉴定、碱性磷酸酶(AKP)染色鉴定及体外分化能力的鉴定。在无人重组白血病抑制因子(hLIF)存在的条件下将ESC悬浮培养4d,形成拟胚体(EB),再经全反式维甲酸(RA)诱导4d,在神经干细胞筛选培养基中扩增,采用免疫细胞化学方法检测NSC特异性标志物Nestin的表达。结果所分离得到的ESC的AKP染色阳性,Oct-4表达阳性,符合ESC的一般特性。ESC经RA初步诱导,NSC选择性培养基筛选培养7d后,形成大量神经球样结构,所形成的神经球样结构呈Nestin抗原阳性。结论体外分离得到的昆明小鼠ESC经RA诱导后,再经选择性培养基筛选培养可获得大量NSC,有望为神经系统损伤及神经变性疾病提供新的治疗途径。

Objective To develop an efficient method by which embryonic stem cells （ESC） can be cultured and induced to differentiate towards neuronal stem cells （NSC） in vitro. Methods Isolated the blastocysts from 3.5d pregnant Kunming mouse. The inner cell mass （ICM） which were obtained following hatch were cultured on the mouse embryonic fibroblast cell （MEF） feeder layer. The stem cells were identified by alkaline phosphatase staining, differentiation ability in vitro and Oct-4 immunocytochemical staining. ESC was subjected to an 8d induction procedure which consisted of 4d of culture as embryonic bodies （EB） without all-trans retinoic acid （RA） and hLIF followed by 4d of culture in the presence of RA. Nestin immunocytochemical staining was performed after amplification in NSC screening medium. Results The isolated cells were positive for Oct-4 and alkaline phosphatase. Therefore the isolated cells presented the general characters of ESC. The ESC which induced by RA was cultivated in NSC screening medium, formed a number of spherical structures. Spherical structures were positive for nestin immunocytochemical staining. Conclusion We obtain large NSC then the ESC which induced by RA were cultivated in NSC screening medium, this result provides a new pathway for healing of nervous system injury and neurodegenerative disease.