一种简单高效的新生鼠海马神经元原代培养方法

A Simple and Effective Primary Culture Method for Newborn Mouse Hippocampal Neurons

【目的】建立一种简单、高效、高纯度的新生小鼠海马神经元的原代培养方法。【方法】取出生24 h内的C57BL/6J新生鼠,分离海马组织,采用胰蛋白酶消化加机械吹打的方法获得单个细胞,用含体积分数1%B27及2%Glutamax-100X、终浓度25μmol/L Glutamate的Neurobasal-A培养基接种培养,3 d后用去Glutamate成分的上述培养基换液,并加阿糖胞苷作用48 h以抑制胶质细胞增长。于倒置相差显微镜下观察细胞的生长状态,采用微管蛋白相关标志物2（MAP2）及Hoechst33258免疫荧光染色鉴定神经元纯度。【结果】此培养方法获得的海马神经元生长状态良好,细胞形态从接种时的圆形透亮、无突起,到培养第7天后发展为神经元胞体聚集、树突发达并形成纵横交错的神经网络;经鉴定,神经元的纯度高达97.2%以上,且能稳定存活2~3周。【结论】该方法操作简单、高效,所得神经元纯度高,结果稳定。

Objective To establish a simple, effective and high-pureity primary culture method tbr newborn mouse hippocampal neurons. Methods C57BL/6J newborn mice within 24 hours after birth were used for the isolation of the hippocampus. Mechanical pipetting combined with enzymic digestion with trypsin was used to obtain monoplast suspension, and then the neurons were planted in Neurobasal-A medium containing volume fraction of 1% B27, 2% Glutamax-100X and 25 μmol/L of Glutamate. After culturing for 3 days, the initial medium for the neurons was totally replaced by the above medium with Glutamate free and with cytosine arabinoside （ Ara-C ） added to inhibit the growth of glial cells for 48 hours. The morphological changes of neuron cells were observed under inverted phase-contrast microscope. Microtubule-associated protein-2 （MAP2） and Hoechst33258 immunofluorescence were used to identify the purity of neurons. Results The growth of hippocampal neurons obtained from the culture method was well on the 7th day of culturing, showing as neuronal soma aggregation, well-developed cytodendrite and formation of criss-crossed nerve net, which were quite different from the round, transparent, and process-free cells at the beginning of planting. The purity of the obtained neurons was more than 95% and the neurons could survive for 2-3 weeks stably. Conclusion The method is simple and effective for obtaining the high-purity and stable neurons.