乳鼠嗅球及皮层神经元的原代培养和Netrin-4受体在神经元定位的初步研究

Primary culture of olfactory bulb and cortical neurons of newborn rat and primary study of the localization of Netrin 4 receptor on the neurons

目的 培养原代嗅球及皮层神经元细胞 ,研究神经轴突生长诱向因子 (Netrin 4 )受体在神经元细胞中的定位。方法 改良Koh法分离、培养出生 12h内Wistar乳鼠的嗅球及皮层神经元 ,采用免疫细胞化学方法鉴定其纯度。碱性磷酸酶 (AP)标记的Netrin 4融合蛋白与原代培养第 7d的神经元孵育 ,以亲和细胞化学法检测Netrin 4受体在神经元的定位。结果 原代培养第 7d的嗅球及皮层神经元胞体饱满 ,突起长并交织成网状 ;免疫细胞化学染色见神经丝蛋白(neurofilament)在神经元胞体及突起中表达 ;嗅球及皮层神经元的细胞膜上均有Netrin 4受体表达。结论 建立起高效、稳定的原代神经元培养方法并应用于神经突起生长诱向因子受体的定位研究。

Objective To establish a primary culture model of olfactory bulb and cortical neurons of newborn rat and investigate the localization of Netrin 4 receptor on the neurons.Methods We used modofied Koh method to cultivate the primary neurons of newborn rat. Evaluation of neurons was made by immunocytochemical techniques. Then, the neurons were incubated with AP4-Netrin 4 fusion protein and localization was performed by in situ staining of AP.Results Cultured neurons were stick to the wall of the cultivation plate when they were growing. The neurons were satiety and with long axon. Immunocytochmical staining showed 90% neurofilament positive staining cell. In situ staining of AP4-Netrin 4 protein bound to neurons showed that Netrin 4 receptor was on the membrane.Conclusion Primary culture of olfactory and cortical neurons of newborn rat by the method is a reliable and highly stable way to obtain neurons. This neuronal culture is a useful model for moleculo-biological and neurobiological studies.