新生SD大鼠皮质神经元的体外培养和鉴定

目的：建立高纯度的新生SD大鼠皮质神经元原代培养方法。方法：取24h内的新生SD大鼠皮质，用木瓜酶和DNaseⅠ共同消化，5％胎牛血清终止消化，吹打分离组织获得单细胞悬液，进行细胞计数，用无血清DMEM／F12种植培养，4h后换成用无血清Neurobasal配制的维持培养液继续培养，尼氏小体染色和免疫荧光法鉴定神经元的纯度。结果：培养第10d，神经元胞体饱满，结构清晰完整，光晕明显，折光性强，可见粗长的树突和轴突，相邻细胞形成紧密网状联系，神经元纯度达到96％以上。结论：经改良和优化，无须添加阿糖胞苷抑制胶质细胞的生长即能够获得生长状态良好、高纯度的神经元。

Cultivation of Cerebral Cortex Neuronal Cells of Newborn SD Rat

Objective： To establish a method for cultivation of cerebral cortex neuronal ceils of newborn SD rat. Methods： The cortexes from newborn（less than 24 h） SD rat were obtained and digested by 2 mg/mL papain and appropriate amount of DNase Ⅰ, terminated digestion with terminating medium and then dissociated into single cell suspension, counted and plated with plating medium. After cultivated in plating medium for 4 h, the neuronal cells were cultured in maintenance medium containing Neurobasal medium, B27 and glutamine. The Nissl＇s staining and indirect immunofluorescence were used to indicate neuronal cells. Results： The neuronal cells on lOth day showed that the body of neuronal cells were full, the structure of neuronal cells were clear and integrity, the axons and the dendrites were thick and long,adjacent cells formed a tight mesh contact. As indicated by indirect immunofluoreseenee using antibodies against neuron specific tubulin βⅢ and Nissl＇s staining, the purity of the neuronal cultures was above 96%. Conclusion： This simplified method（added DNase Ⅰ, serum-free medium and cytosine-arabinofuranoside-free） is cost-effective for primary culture of cerebral cortex neuronal cells and the neurons obtained showed high uniformity, purity and long-term viability.