C57BL/6胎鼠大脑皮质神经元体外培养与鉴定

Primary culture and identification of neurons from embryo cerebral cortex of C57BL/6 mice

关键词:  C57BL  6  胚胎  大脑皮质  神经元  免疫荧光

Key words:  C57BL/6  embryo  cerebral cortex  neurons  fluorescent antibody technique

摘要:

目的探讨近交系C57BL/6胎鼠皮质神经元体外培养方法，观察其生长情况，并进行鉴定。方法分离孕17dC57BL/6胎鼠大脑皮质，剪碎吹打，静置取上清液，余组织块以木瓜酶和DNaseI消化，差速贴壁提纯，培养6h后以添加含谷氨酰胺和B27的Neurobasal培养基继续培养，观察其生长情况，并于体外培养3、5d时，以神经元特异性核蛋白（neuronspecificnuclearprotein，NeuN）和微管相关蛋白一2（microtubule-associatedprotein-2，MAP-2）为标志物，用免疫荧光双染法对分离培养的原代细胞进行鉴定。结果体外培养6h细胞已贴壁，少量细胞发出l~3个短小突起；此后神经元相继发出突起、并伸长，与邻近细胞突起互相接触，培养5d突起已互相交织成网；体外培养3、5d的NeuN阳性细胞率分别为（94.1±5.0）%、（95.0±5.6）%；MAP一2阳性细胞率分别为（96.7±2.9）％、（95.6±5.6）％，NeuN、MAP-2阳性细胞率差异无统计学意义（P〉0.05），NeuN和MAP-2鉴定结果一致性好。结论本方法制备的C57BL/6胎鼠皮质原代神经元纯度高，损伤小，生长发育状态良好，无明显增殖性，可作为神经元体外研究的良好实验材料。

Abstract:

Objective To establish a method of isolation and primary culture of neurons from the embryo cerebral cortex of C57BL/6 mice. Methods The cortices of El7 C57BL/6 embryos were isolated, minced and triturated. The supernatant was collected and the remaining tissues were digested with papain and DNaseI. The cells collected were purified by differential adhesion method. After 6 h of incubation, the cells were cultured in Neurobasal medium supplemented with glutamine and 2% B27. The neurons were observed at different stages and identified by NeuN/MAP-2 double fluorescence staining after 3 and 5d of incubation. Results After 6 h of incubation 1-3 small processes were observed in a few neurons, the processes were prolonged and connected to adjacent neurons to form network on d5. The rates of NeuN-positive neurons were （94.1±5.0）% and （95.0±5.6 ） %on d3 and d5, respectively （P＞0.05 ）; and the rates of MAP-2-positive neurons were （96.7±2.9）% and （95.6±5.6）%, respectively （P＞0.05 ）. Conclusion The method of primary culture established in this study can stably supply high-quality cortical neurons from C57BL/6 embryos, which can be used for in vitro neuronal research.