大鼠胚胎脊髓神经干细胞的分离培养及其向神经细胞的诱导分化

In vitro culture and induced neuronal differentiation of rat embryonic spinal cord-derived neural stem cells

目的分离培养大鼠胚胎脊髓神经干细胞(SNSCs),研究其体外增殖分化的特点,模拟大鼠胚胎时期脊髓发育的微环境诱导SNSCs向神经细胞定向分化。方法显微机械分离孕10～11d胚胎大鼠脊髓神经干细胞,无血清悬浮培养,以复合添加剂N4定向诱导分化,并拍照记录不同时期细胞分化的形态;用不同荧光剂进行细胞免疫荧光染色,并计算阳性细胞的平均分化比例。结果显微机械分离SNSCs方法,所分离的SNSCs具有较高的增殖能力,经复合添加剂N4诱导可以分化为神经元、星型胶质细胞、少突胶质细胞。结论采用显微机械分离SNSCs方法简单、效率高,N4添加剂可以高效诱导SNSCs分化为神经细胞,且分化的细胞中神经元比例高。

Objective To induce directional differentiation from spinal cord-derived neural stem cells （SNSCs） into neurocytes in the simulated microenvironment of development of embryonic spinal cord of rat after isolation and culture of SNSCs and to investigate the proliferation and differentiation of SNSCs in vitro. Methods SNSCs were isolated mechanically from rat embryonic （E10 to 11d） spinal cord under microscope. SNSCs were cultured and maintained in serum-free medium. Directional differentiation from SNSCs to neurocytes was induced by adding N4 supplement. The morphologic features of the differentiated cells were noted. Cultured and differentiated cells were identified by immunochemistry stain and the mean differentiating percentages of the positive cells were calculated. Results SNSCs isolated by the mechanical method under microscope were vigorous and proliferative, and could differentiate into neurons, astrocytes and oligodendrocytes. Conclusion The mechanical method under microscope of isolating SNSCs is simple and efficient to obtain a high percentage of neurons. N4 supplement can induce SNSCs to differentiate into neurocytes.