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目的对原有大鼠海马神经元原代培养方法进行改进,在获得数量多、纯度高、体外生长时间长的神经细胞的同时,简化操作流程、节省时间、减少污染。方法分离24h内出生的Wistar大鼠双侧海马,采用机械吹打法,以沉降法代替过滤制备单细胞悬液,细胞接种24h后培养液更换为添加B27的DMEM/F12无血清培养基,以后每周半量换液2～3次。用倒置相差显微镜观察细胞形态,利用神经元特异性标志物神经元特异烯醇化酶(NSE)鉴定神经细胞的纯度,并以钙影像仪测定KCl作用神经元后细胞的钙离子浓度变化,了解神经元的兴奋性。结果此简易方法节约操作时间及科研成本,减少了污染的机会。所培养的神经元杂质少、纯度高、细胞活性好,体外存活时间长。结论该方法简便可行,结果稳定,用该方法培养的大鼠海马神经元可作为神经元体外培养的良好实验模型。
OBJECTIVE: To improve the method of primary culture of primary cultured rat hippocampal neurons, and to obtain a large number of neurons with high purity and prolonged growth in vitro, while simplifying procedures, saving time and reducing pollution. Methods The bilateral hippocampus of Wistar rats born within 24 hours were isolated by mechanical blowing method and the single cell suspension was prepared by sedimentation instead of filtration. After 24 hours, the medium was changed to DMEM / F12 serum-free medium supplemented with B27 Change the amount of liquid half a week 2 to 3 times. The cell morphology was observed by inverted phase contrast microscope. The neuron-specific enolase (NSE) was used to identify the purity of nerve cells. The change of calcium concentration in KCl-treated neurons was detected by calcium imaging. Excitability of the yuan. The result of this simple method saves operating time and research costs, reducing the chance of contamination. Neurons cultured by less impurities, high purity, good cell activity, long survival in vitro. Conclusion The method is simple and feasible, the result is stable. The hippocampal neurons cultured by this method can be used as a good experimental model of neuron culture in vitro.