新生大鼠视皮层神经元的原代培养及鉴定

Primary cultivation and identification of visual cortical neuron in neonate rat

目的探讨新生大鼠视皮层神经元的原代培养方法,以获取数量多、纯度高的视皮层神经元。方法取新生1d内的SD大鼠视皮层,通过胰酶消化法获取单细胞悬液,倒置相差显微镜下计数后种植于6孔培养板内培养。培养24h后用终浓度10μmol·L-1阿糖胞苷处理,抑制非神经元的生长,作用24h后半量换液,以后每隔1d半量换液1次。每天倒置相差显微镜下观察细胞的生长状况和形态变化,Nissl染色法进行视皮层神经元鉴定,免疫荧光显示神经元特异性烯醇化酶鉴定培养视皮层神经元的纯度。结果接种4h后,大部分细胞已贴壁,细胞立体感较强;接种后24h,大部分细胞长出短的突起,细胞形态发生变化,胞体呈多边形或三角形,细胞周围有或多或少的光晕;培养4d后,细胞突起伸长、增粗;培养6～8d后,细胞状态较佳,细胞胞体饱满,光晕明显,突起进一步伸长、增粗,且分支多,突起交织成网状,非神经元细胞减少,为实验的最佳时间;培养14d后,细胞开始退化。培养6d行焦油紫染后,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,原代培养的细胞中,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。结论本研究获得视皮层神经元的方法简单经济,通过形态学观察、Nissl染色及免疫荧光染色可以对体外培养的视皮层神经元细胞进行准确鉴定。

Objective To explore-the primary cultivation method of cortical neu- ron in neonate rat for obtaining more neuron with high purity. Methods The cerebral cortex of SD rats with postnatal one day was received, and the monoplast suspension was obtained by enzyme digestion, and then cultured in six orifices after being calculat- ed under inverted phase contrast microscope. After being cultured for 24 hours, the cells were treated with 10 μmol · L-1 cytarabine,the growth of negative neuron was inhibi- ted,half of the liquid was replaced after 24 hours, and then did it 1 time 1 day. The growth and morphologlc changes of cells were observed under inverted phase contrast microscope, the neurons was identified by Nissl staining, and the NSE was displayed by immunofluorescence to indentify the purity of neuron. Results After being cultured, most neuron adhered with strongly three-dimensional round morphous at 4 hours;short dendrites appeared in most neuron and the morphous changed with round or oval ap- pearance at 24 hours;the dendrites prolonged and thickened with many branch at 4 days ;the neuron was with well-stacked cell body, clear halation formed, the dendrites further prolonged and thickened with many branches which formed the integrated neu- ral network, negative neuron decreased from 6 days to 8 days, and it was the best cul- tured time;then the neuron began to degenerate at 14 days. The Nissl body appeared he- patic particle and plaque stained by cresyl violet at 6 days after culture and obeserved under inverted phase contrast microscope. The fluorescence staining showed most neu- ron were stained in green with well morphous and clear nuclear, and the rate of neuron in all cells was high. Conclusion The method used in this study is simple and eco- nomical for culturing cortical neuron,which can be accurately indentified through mor- phous observation, Nissl staining and fluorescence staining.