新生大鼠离体海马神经元原代培养方法及鉴定

Primary culture and identify of mature rat neonatal rat hippocamal neurons in vitro

目的:建立一种稳定的生后大鼠离体海马神经元的培养方法。方法:无菌环境下将出生24 h内SD大鼠断头取脑分离海马,经消化后差速贴壁,采用无血清培养基进行培养,倒置显微镜下观察不同时间段细胞生长形态变化:采用Hoechst33258与NeuN抗体双染方法鉴定神经元。结果:采用差速贴壁后,使用无血清培养基培养的神经元生长良好,纯度较高,12 d时经鉴定神经元纯度可以达到92%以上。结论:差速离心法后可以采用无血清培养神经元,培养的神经元具有纯度高,结果稳定的优点,该方法为以后进行相关的研究奠定了基础。

Objective： To establish a stable cuhureal method of neonatal rat hippocamal neurons in vitro. Methods： Isolated hippocampu of brains of SD rats born within 24 h after guillotine under sterile condition, the hippocampus was disgested, then the cells were differential speed adherent inoculated seeded in a plates inoculated poly-L-lysine and cul- tured in serum - free medium. The morphological change of neurons different developmental and differential stages was ob- served under phase-contrast microscope and the neurons were identified by Hoeehst33258 and neuron-specific anti-NeuN antibody double staining method. Results： Differential speed adherent mothod and cultured neurons by serum-free medi- um, the hippoeamal neurons showed good grow and high purity （ mature neurons purity rate can reach more than 92% ）. Conclusion： With the method of differential attachment and cultured neurons by serum-free medium, the mature neurons showed the advantages of high purity and results stable. This laid the foundation for future related research.