一种改进的大鼠皮层神经元原代培养方法及其性质鉴定

目的：对原有大鼠皮层神经元原代培养方法进行改进, 以获得数量更多、纯度更高、体外生长时间更长的神经细胞进行相关实验研究。方法: 使用3种不同成分的液体对培养器皿进行序贯预处理, 分离16~17 d胎龄的Wistar大鼠胎鼠皮层, 采用木瓜蛋白酶化学消化与机械吹打相结合的方法制备单细胞悬液, 经细胞计数后按照不同实验目的进行梯度密度接种。细胞接种当日4~6 h将含有血清的接种培养液换为添加B27的Neurobasal无血清培养基, 培养第3天(DIV3)用终浓度10 μmol/L的阿糖胞苷(cytosine arabinoside, Ara-C)处理24 h抑制胶质细胞增殖, 以后每周半量换液1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微管相关蛋白2(microtubule associated protein 2, MAP2)与细胞核双标记法鉴定培养神经细胞的纯度, 并以免疫荧光法检测突触前、后标记物以评估突触形成情况。 结果: 与单纯多聚赖氨酸预处理培养器皿, 胰蛋白酶化学消化法分离细胞及含血清培养基培养的原方法相比, 方法改进以后收获的细胞产量明显增加, 且消化过程对细胞损伤小, 接种后细胞分散均匀, 杂质少, 纯度高, 树突、突触发育正常, 可长期存活。 结论: 该方法简便可行, 结果稳定,用该方法培养的大鼠皮层神经元可作为神经元体外培养的良好实验模型。

An improved method for primary culture of rat cortical neuron and cell identification

Objective:To improve previous method of primary rat cortical neuron culture to get purer and more long-lasting cells for study.Methods:Timed-pregnant Wistar rats at a gestational age of 16 or 17 days(16-17 d) were used.Fetal brains were removed and the cerebral cortices were dissected out.Papain digestion and mechanical dissociation were combined to conduct mono-cell suspending media.Four to six hours(4-6 h) post-plating,all plating media were removed from cultures and replaced with Neurobasal medium supple...