摘要:
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。
Abstract:
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.
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