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

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17 d C57BL/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6 h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、5 d时,以神经元特异性核蛋白(neuron specific nuclear protein,NeuN)和微管相关蛋白-2(microtubule-associated protein-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6 h细胞已贴壁,少量细胞发出1～3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5 d突起已互相交织成网;体外培养3、5 d的NeuN阳性细胞率分别为(94.1±5.0)%、(95.0±5.6)%;MAP-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。

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.