体外分离培养Balb/c小鼠胚胎干细胞向神经细胞定向诱导分化

目的: 探讨体外分离培养、诱导胚胎干细胞(ESCs)分化为神经细胞的高效方法. 方法: 自孕3.5 d的Balb/c小鼠获得附植前的早期胚胎,机械剥离法去除胚胎的滋养层细胞获取内细胞团(ICM)分别在小鼠胚胎成纤维细胞(MEF)饲养层上和明胶包被的培养板中培养获得类ESCs克隆. 进行碱性磷酸酶染色,SSEA-1细胞免疫化学鉴定及体内分化实验鉴定. 模拟体内神经系统发育过程采用序贯诱导方法定向诱导ESCs分化为神经细胞,进行分化后细胞的免疫细胞化学鉴定. 结果: 所分离得到的细胞碱性磷酸酶染色阳性,SSEA-1表达阳性,体内分化实验证实具有多向分化潜能. 符合ESCs的一般特性. 经过序贯诱导方法可以高效诱导ESCs分化为神经细胞. 结论: 体外分离得到的Balb/c小鼠的ESCs经过模拟体内神经系统发育过程经过序贯诱导方法可以高效的诱导为神经细胞,且所获得的神经元比例高.

In vitro differentiation of embryonic stem cells from Balb/c mouse into neurocytes

AIM: To develop an efficient method by which embryonic stem cells(ESCs) can be cultured and induced to differentiate towards neurocytes in vitro. METHODS: Isolate the blastula from 3.5 d preqnant Balb/c mouse. The inner cell mass (ICM) which were obtained following the stripping of trophoblast by mechanical method were cultured on the mouse embryonic fibroblaste cell (MEF) feeder layer or 0.1% gelatin coated dishes. The stem cells were identified by alkaline phosphatase staining, differential potency in vivo and SSEA-1 immunocytochemical staining. The isolated cells were induced to differentiate towards neurocytes by sequence induction that mimicking the intrinsic developmental process of the neural system. RESULTS: The isolated cells were positive for alkaline phosphatatse and SSEA-1 (stage specific embryonic antigen 1). Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated cells presented the general characters of ESCs. Then the isolated cells were able to differentiate into neurocytes in vitro with the help of sequence induction. CONCLUSION: Under the sequence induction that mimicking the intrinsic developmental process of the neural system, ESCs inolated form Balb/c mouse can differentiat into neurocytes with a high proportion of neurons.