海马神经元原代培养与纯度鉴定方法的优化

Optimization Methods About Primary Culture of Hippocampal Neurons and Purity Identification

目的:通过优化和改进传统的神经元培养方法,探讨一个简单、高纯度的大鼠胚胎海马神经元的原代培养及活性检测方法。方法:颈椎脱臼处死孕鼠,迅速取海马,剥离血管网等结缔组织后,消化并吹打制成细胞悬液,以适当的密度接种入DMEM/HG培养基,4h后换用Neurobasal培养基,以后每2-3d进行1/3量换液。倒置显微镜观察神经元的形态变化,并采用CCK-8试剂盒检测神经元活性和神经元特异性烯醇化酶(NSE)鉴定神经元纯度。结果:细胞接种4h后贴壁,随着培养时间的延长神经元的形态发生一系列变化,从圆形、透亮、体积小逐渐变为椭圆形、三角形或椎形,有光晕,胞体增大,突起伸长且分支增多,在培养第4天后逐渐连接呈网络状。神经元纯度随时间的延长逐渐升高,培养第7天纯度高达90%以上。神经元在培养的第9、10天达到成熟,即可用于后续研究。结论:本实验方法简便易行,神经元纯度高、细胞活性好,体外存活时间长,是体外研究的良好实验模型,值得推广应用。

Objective： To explore a simple and high-purity method about primary culture of rat embryon- ic hippocampal neurons, through optimization and improvement of traditional neuron culture. Methods： Pregnant rats were sacrificed by cervical dislocation, and the rat embryonic hippocampal tissue was taken out quickly. The hippocampal neurons were separated, digested and made into cell suspension, and then inoculated into DMEM/HG medium at an appropriate density. DMEM/ HG medium were replaced by neurobasal medium after 4 hours. Thereafter, one third volume of culture medium was refreshed every two or three days. The neurons ~ growth and morphological changes were observed with inverted microscope, neurons activity was detected with CCK 8 Kit, and the purity was identified with neuron-specific enolase （NSE）. Results： Hippocampal neurons had adhered after being cultured for 4 hours. Neurons had a series of morphologic changes with extending of incubation time. The purity of neurons increased gradually with extending culture time, and it was over 90 percent on the 7th day. Neurons were matured on the 9th or 10th dayand were used for the follow-up studies. Conclusion： acquire high purity of neurons, cell activity is good, worth being applied widely in vitro studies as a good The serum free culture method is simple to and cell survival time is long in vitro. It is cell model.