原代神经元与星形胶质细胞的混合培养方法..pdf

72 生理学报Acta Physiologica Sinica , February 25, 2013, 65(1): 72–76 实验技术 原代神经元与星形胶质细胞的混合培养方法 * 石 莹，周密，姜民 复旦大学脑科学研究院，医学神经生物学国家重点实验室，上海 200032 摘 要：原代培养的海马神经元是研究神经细胞中蛋白运输与亚细胞空间定位的有效研究工具。本文旨在建立海马神经元与 皮层胶质细胞混合培养的方法，以期提供状态良好的神经元供研究用。新生2~3 d Sprague-Dawley (SD)大鼠断头处死，从大 2~3 25 cm2 脑半球表面片取 片皮层组织，切碎后用胰蛋白酶消化成单细胞悬液，种植于 培养瓶内。种植后第四天，采用剧烈 敲击培养瓶的方法去除非星形胶质细胞的杂质细胞，继续培养贴壁细胞直至细胞接近铺满瓶底，改用含阿糖胞苷的神经细 胞培养液。原代培养的大鼠海马神经元种植在这种预先培养好的皮层星形胶质细胞层上。镜下观察结果显示，本方法培养 的原代星形胶质细胞纯度较高，共培养的神经元状态较佳，存活时间长，突触前结构发育良好，可以耐受转染操作。以上 结果提示，本文所建立的原代神经元与星形胶质细胞的混合培养方法操作简单，能够可靠地培养出健康的神经元。 关键词：神经元；胶质细胞；共培养 中图分类号：Q7 A protocol for primary dissociated astrocyte and neuron co-culture SHI Ying, ZHOU Mi, JIANG Min* Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, China Abstract: Cultured primary hippocampal neurons are ideal tool for investigating the subcellular localization and trafficking of neu- ronal proteins. The aim of the present study was to establish a method to co-culture hippocampal neurons and cortical astrocytes, which would guarantee well conditions of neurons. Newborn Sprague-Dawley (SD) rats were sacrificed by decapitation. The cortex of cerebrum was cut into pieces, and the cortical tissue was digested with trypsin. The liquid suspension of single cells was planted onto a 25 cm2 culture flask. On the fourth day of culture, the tissue cells except astrocytes were removed by intensive agitation of culture flask. Purified astrocytes were allowed to grow continuously until they reached most area of flask. At this time point, we replaced the culture media with neuronal cell media containing cytarabine, and planted the primary culture of rat hippocampal neurons o