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目的建立新生大鼠大脑皮质神经元的原代培养方法,以获取数量多、纯度高的神经元。方法取出生1 d内的SD大鼠的大脑皮质,通过胰酶消化法获取单细胞悬液,计数后种植于6孔板培养,培养24 h后用终浓度10μmol.L-1的阿糖胞苷处理。采用倒置相差显微镜每天观察细胞的生长状况和形态变化,Nissl’s染色进行神经元的鉴定,免疫荧光显示神经元特异性烯醇化酶鉴定培养神经元的纯度。结果培养2 h后,大部分细胞已贴壁,细胞圆且立体感较强;接种24 h后,大部分细胞长出短的突起,细胞形态发生变化,胞体呈圆形或椭圆形;培养4 d后,细胞突起伸长、增粗,且分支多,突起交织成网状;培养6～8 d细胞状态最佳,细胞胞体饱满,光晕明显,形成完整神经网络;培养14 d后,细胞开始退化。培养6 d行焦油紫染色后,倒置相差显微镜下观察,可见细胞内的尼氏体呈蓝紫色的颗粒或斑块。免疫荧光染色结果显示,大部分细胞为绿染细胞,形态良好,核大而清晰,神经元所占比例高。结论本研究获得皮质神经元的方法简单经济,通过形态学观察、Nissl’s染色及免疫荧光染色可以对体外培养的皮质神经元细胞进行准确鉴定。
OBJECTIVE: To establish a primary culture method of cerebral cortex neurons in neonatal rats to obtain a large number of neurons with high purity. Methods The cerebral cortex of SD rats was taken out and the single cell suspension was obtained by trypsin digestion. After counting, the cells were seeded in 6-well plates and cultured for 24 h. Cells were treated with 10 μmol·L-1 Ara-C Glycoside treatment. The growth and morphological changes of the cells were observed daily by inverted phase contrast microscope. Nissl’s staining was used to identify neurons. Immunofluorescence showed neuron-specific enolase was used to identify the purity of cultured neurons. Results After cultured for 2 h, most of the cells were adherent and had a round and three-dimensional appearance. After 24 h of inoculation, most of the cells grew short protuberances and the morphology of the cells changed. The cell bodies were round or oval. After 6 days, the cell morphology was best, the cell body was full and the halo was obvious, forming a complete neural network. After 14 days of culture, the number of cells Start to degenerate. After cultured for 6 days, the tar violet staining was performed and the inverted phase contrast microscope was used to observe that the Nissl bodies in the cells were blue-purple granules or plaques. Immunofluorescence staining showed that most of the cells were green-stained cells with good morphology, large and clear nucleus and high proportion of neurons. Conclusion The method of obtaining cortical neurons in this study is simple and economical. Nissl’s staining and immunofluorescence staining can accurately identify cultured cortical neurons in vitro.