胚胎大鼠神经干细胞的分离培养及其基本属性的观察

Observation of Isolation,Cultivation and Characteristics of Neural Stem Cells of Fetal Rats

目的分别以单纯机械吹打法和胰蛋白酶消化法分离培养神经干细胞(NSCs),并观察NSCs的生长、增殖和分化特点。方法采用无血清培养技术从胎鼠的大脑皮层和海马通过单纯机械吹打和胰蛋白酶消化分离培养原代细胞,并加血清诱导其分化,借助免疫细胞化学技术检测培养的神经干细胞及其分化后神经元和星形胶质细胞标志蛋白的表达;并应用相差显微镜观察神经干细胞的生长特点及分化后的细胞形态学变化。结果①从胎鼠大脑皮层及海马能分离培养出具有很强增殖力的细胞,它们能稳定表达巢蛋白,经诱导分化后可表达神经元和星形胶质细胞的特异性抗原。②与用单纯机械吹打分离细胞法相比,用胰蛋白酶消化后,在神经干细胞培养基中有很少或几乎没有未分离的组织团块,原代细胞背景更清晰。结论①应用胰蛋白酶消化,能分离培养出纯度较高的神经干细胞。②从大鼠大脑皮层及海马能成功地分离、培养出神经干细胞,它是进一步研究神经干细胞的良好材料。

Objective：Isolating and cultivating neural stem cells（NSCs）by triturating only with glass pipette and by digestion of trypsin and observing the features of their growth,proliferation and differentiation.Methods：The primary cells dissociated by both methods from the cerebral cortex and hippocampus of fetal rats were cultured with serum-free incubation technique and differentiation were induced with serum.Immunocytochemical study was carried out to verify NSCs,neurons and astrocytes.The growing features of NSCs,as well as their morphological changes after differentiation,were observed under phase contrast microscope.Results：（1）The nestin-positive NSCs derived from the cerebral cortex and hippocampus have proliferating potential.The differentiated cells expressed specific markers of neuron and astrocyte respectively.（2）Compared with triturating only with glass pipette,there were few or not almost histio-masses and clearer background in NSCs culture medium after digestion of trypsin,but the growth of NSCs has not significant difference between them.Conclusion：（1）We obtained NSCs with higher purity by digestion of trypsin.（2）The brains of fetal rats are proper candidate for further study of NSCs.