新生大鼠小脑颗粒神经元原代培养与鉴定

Primary culture and identification of cerebellar granule neurons from newborn rats

目的:建立一种较为理想的小脑颗粒神经元原代培养方法。方法:取新生5～7天SD大鼠,分离小脑皮质,胰酶消化后差速贴壁,种植在预先涂有左旋多聚赖氨酸的培养板内,第3天加入阿糖胞苷纯化神经元;采用神经元特异性烯醇化酶免疫细胞荧光技术鉴定神经元。结果:细胞存活率达(98±1.07)%;24 h内基本贴壁;第3天细胞突起增多、变长;培养6～8天,细胞突起交织成网,形成典型的神经细胞网络;神经元特异性烯醇化酶鉴定神经元细胞占90%左右。结论:实验获取神经元纯度较高,是小脑颗粒神经元体外培养的一种较理想的方法。

Objective：To establish a suitable primary culture method of rat cerebellar granule neurons.Methods：Rat cerebellar granule neurons were prepared from 5-7 day old Sprague-Dawley rat pups,the cerebella was freed of meninges,minced,trypsinized,then the cell suspension was preplated for 30 min for remove any glial cells,dissociated cells were seeded at plates which had been pre-coated with Poly-L-Lysine,arabinosylcytosine was added to the culture medium on day 3 after seeding for inhibition of non-neuronal cell division.Neurons were identified by neuron-specific enolase immunofluorescence technic.Results：The survival rate of the cells was（98±1.07）%;the neurons were affixed to the culture plate after 24 hours,neurite growth was apparently on day 3,integrated neural network was formed on day 6-8.Cerebellar granule neurons was about 90% by neuron-specific enolase identifying.Conclusions：Neuron purity was higher in the experiment;it is a perfect technique for primary culture of rat cerebellar granule neurons.