大鼠大脑皮质神经元原代培养不同培养体系及纯化方法的比较研究

Comparative study on cortical neuron primary culture system and purification in two groups of rats

目的:比较两种大鼠大脑皮质神经元的培养及纯化方法不同组合的优劣,探讨科学可靠的神经元培养纯化方法。方法:采用DMEM/F12+血清的有血清培养体系和神经基础培养基(Neurobasal)+B27的无血清培养体系培养原代神经元。在接种3天后分别采用加入阿糖胞苷和5-氟脱氧尿嘧啶两种方法进行神经元的纯化。在1天、3天、7天采用形态学及7天时免疫荧光化学法鉴定神经元的纯度及生长状态。结果:1天、3天时,有血清培养体系和无血清体系中的神经元的形态结构和密度无明显差别,在分别加入阿糖胞苷和5-氟脱氧尿嘧啶后,7天时无血清体系中加阿糖胞苷组细胞密度明显低于其他三组细胞密度。通过免疫荧光化学法鉴定可见无血清体系中的神经元纯度明显高于有血清体系。结论:有血清体系的神经元纯度较差,阿糖胞苷和5-氟脱氧尿嘧啶不能显著抑制胶质细胞等杂细胞的生长。无血清培养体系加阿糖胞苷的神经元细胞受损较多,无血清培养体系加5-氟脱氧尿嘧啶纯化培养的大鼠大脑皮质神经元,纯度较高,细胞数量合适,是体外研究神经系统相关理论的良好模型。

Objective： To investigate the reliable method of neuron primary culture and purification by comparing the different methods of culture and purification. Methods： Neuron cell of SD rats were separated and cultured in DMEM/F12 serum culture system and Neurobasal/b27 free serum culture system. After culture 3 days, adding arabinosylcytosin and 5- fluorodeoxyuridine to purify neuron cell. To observe the morphology of the neuron cells on lday,3day and 7day by inverted microscope and observe the purity quotient and growth state of neuron cell on 7day by immunofluorescence. Results： morphosis and cell density of neuron cells have no different on lday and 3day, but cell density of neuron cell by arabinosylcytosin cells is lower than other on 7day, purity quotient of neuron cells in free serum culture system is better than it in serum culture system by immunofluorescence.adding arabinosylcytosin have more influence on growth state of neuron cell than by 5- fluorodeoxyuridine. Conclusion： free serum culture system with arabinosylcytosin is a better method of culture and purify neuron cell.