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目的建立一种简单、稳定的乳鼠海马神经元的分离及原代培养方法。方法选用出生24 h内的乳鼠,分离双侧海马区,机械吹打分散,获得细胞悬液。用含胎牛血清的DMEM/F12培养基培养24 h后,换成添加B27的Neuralbasal培养基进行原代培养,第7天采用神经元特异性烯醇化酶(NSE)免疫荧光细胞化学染色鉴定神经元纯度。结果在体外培养条件下海马神经元细胞结构特征明显,能形成典型的神经细胞网络,100%细胞呈NSE阳性。结论依靠简单的机械分散并配合特定培养条件可获得生长状态良好、纯度高的海马神经元。
Objective To establish a simple and stable method for the isolation and primary culture of neonatal rat hippocampal neurons. Methods Neonatal rats were born within 24 h, bilateral hippocampus were isolated, mechanically dispersed and dispersed to obtain cell suspension. After cultured for 24 h in DMEM / F12 medium containing fetal bovine serum, the cells were cultured in Neuralbasal medium supplemented with B27, and neurons were identified by immunofluorescence cytochemical staining on the 7th day with neuron-specific enolase (NSE) Yuan purity. Results Under the culture conditions of hippocampal neurons, the cell structure of the hippocampus was obvious, forming a typical network of neural cells, with 100% NSE positive. Conclusions Hippocampal neurons with good growth status and high purity can be obtained by simple mechanical dispersion combined with specific culture conditions.