新生大鼠大脑皮质神经元的原代培养及其鉴定

Primary Cultivation and Identification of Cortical Neuron in Neonatal Rats

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 the primary cultured method of cortical neuron in neonate rats for obtaining more neuron with high purity. Methods The cerebral cortex of SD rat with postnatal one day was received,and the monoplast suspension was obtained by enzyme digestion and then cultured in six orifices after calculated under inverted phase contrast microscope.After cultured 24 hours,the cells were treated by 10 μmol·L-1 cytarabine.The growth and morphologic change of cells were observed under inverted phase contrast microscope,the neurons were identified by Nissl′s staining,and the theneuronspecific enolase was displayed by immunofluorescence to indentify the purity of neuron. Results After culture,most neuron adhered with strongly three-dimensional round morphous at 2 hours,short dendrites appeared in most neuron and the morphous changed with round or oval appearance at 24 hours,the dendrites prolonged and thickened with many branch at 4 days,the neuron with well-stacked cell body and obvious halation formed the integrated neural network from 6 days to 8 days,then the neuron began to degenerate at 14 days.The Nissl body were hepatic particle and plaque stained by cresyl violet at 6 days after culture under inverted phase contrast microscope.The fluorescence staining showed that most neuron were stained in green with well morphous and clear nuclear,the rate of neuron in all cells was high. Conclusion The method used in this study is simple and economically for culturing cortical neuron,which can be accurately indentified through morphous observation,Nissl′s staining and fluorescence staining.