BACKGROUND:Primary culture in vitro of neurons plays an important role in the development, regeneration, signal transduction mechanisms, neuropharmacology and gene expressions of the nervous system. OBJECTIVE:To establish a simple method for primary culture of high-purity cortical neurons in neonatal Sprague-Dawley rats. METHODS:Cortical tissues were acquired from neonatal Sprague-Dawley rats born 1 day. In traditional experimental group, the whole cortex was removed;in improved experimental group, the cortical tissues, 2-3 mm thick on the brain surface were removed. Single cel suspensions were prepared after papain digestion and centrifugation and were then seeded onto 24-wel culture plates containing neuron solutions for primary culture (1×105 per wel ). Cel s were identified by neuronal specific markers MAP-2 and Tuj1 after 3-day culture. The number of neurons and neurite length were observed under inverted phase contrast microscope and recorded at 6, 24, 48 and 72 hours, 5 and 7 days of culture, resprctively. RESULTS AND CONCLUSION:The cultured cel s expressing MAP-2 and Tuj1 were neurons that could be used in the fol owing experiments. The purity of neurons in the improved experimental group was 92%at 3 days, while only 51%in the traditional experimental group. Cel s in both two groups had attached to the wal presenting with smal processes at 6 hours, and a simple neural network formed at the 3rd day until dense neural networks could be found at the 5th day. To conclude, our culture method herein is simple and convenient, and can be used to produce neurons with high purity, which wil be helpful for the experimental studies on cortical neurons from Sprague-Dawley rats.%背景：神经元的体外原代培养在研究神经系统的发育、再生、信号转导机制、神经药理学以及基因表达方面具有极其重要的地位。 目的：建立一种操作更加简单且能够得到较高纯度新生SD大鼠皮质神经元原代培养的方法。 方法：取1 d新生SD大鼠皮质。传统方法实验组：取整个皮质；改良方法实验组：取SD大鼠表面2.0-3.0 mm处皮质。木瓜蛋白酶消化后离心，制备成单细胞悬液，以1×105/孔的浓度接种至含神经元培养液的24孔培养板进行原代培养。培养3 d时采用免疫细胞化学染色方法，使用神经元特异性标志物Tuj1与MAP-2双标记法鉴定所培养的细胞；采用倒置相差显微镜观察6，24，48，72 h及5，7d细胞数和突起长度并记录。 结果与结论：①所培养的细胞可表达神经元特异性标志物Tuj1与MAP-2，因此所培养细胞为神经元，可用于之后实验；②实验组培养至第3天时神经元的纯度已达到峰值92%，而普通实验组神经元的纯度为51%；③2组细胞培养至6 h均已贴壁且长处小突起，培养至第3天时，细胞已初步形成神经网络，培养至5 d时神经网络密集；④研究所用方法简便能够稳定的培养出纯度较高的神经元，可以用于SD大鼠皮质源性神经元的相关实验研究。
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