大鼠胚鼠皮层神经元培养与鉴定

Primary Culture and Identification of Cortical Neuron from Rat Embryonic Mouse

目的：体外建立一种高纯度的大鼠皮层神经元原代培养的方法。方法：孕15~18dSD大鼠胚鼠为研究对象,分离胚鼠大脑皮层,采用胰酶消化方法与机械吹打相结合的方法制备细胞悬液,以适当的密度接种于培养板;细胞培养过程中,以含10%FBS的高糖DMEM/F12培养液接种细胞,24h后全量换液换为含2%B27的Neurobasal-A培养液,抑制非神经元细胞的增殖,以代替阿糖胞苷继续维持培养,倒置显微镜下定时观察细胞形态。结果：神经元细胞分散均匀,紧密贴壁,生长状态良好,神经细胞周围突起相互连接形成神经网络,通过免疫细胞化学方法鉴定神经元纯度为（93.7±5.5）%。结论：此实验方法体外可获得较高纯度的大鼠皮层神经元细胞。

Objective： Establish a method of primary culture high-purity rats cortical neuron in vitro. Methods： The pregnant 15-18d SD rat embryonic mouse was the object of study, which cerebral cortex was stripped. The cell sus- pension was prepared by combining mechanical percussion method with trypsin digestion method. Cells with the appro- priate density were inoculated in the culture plate. The just inoculated cells were cultured in the high glucose DMEM/ F12 medium containing 10% FBS. The medium was exchanged wholy for Neurobasal-A medium containing 2% B27 af- ter 24h,which restrained the proliferation of non-neuronal cells and substituted cytarabine to maintain culture cells. Cell morphology was observed regularly under the inverted microscope. Results： Neurons were distributed evenly, tightly ad- herent, and grew well. Nerve cell axons and neurite were connected to each other to form a neural network. Neurons purity was identified （93. 7 ±5. 5）% by immunocytochemical method. Conclusion： Higher-purity rat cortical neuron cells were successfully cultured in vitro with this experimental method.