陈学周++毕意辉++张可可++张明凯++尤涛

摘 要：目的：通过对新生啮齿类海马神经元的分离和培养，尝试建立一个简单、稳定、高效的啮齿类海马神经元原代培养方法，为脊髓损伤的相关分子机制研究提供目的细胞。方法：取新生SD乳鼠的海马组织，通过低浓度胰酶消化制成细胞悬液、4h差速贴壁后使用无血清Neurobasal培养基培养，倒置显微镜观察细胞生长状态，免疫荧光对海马神经元相关微管蛋白-2（MAP2）行特异性染色，结合DAPI核染色鉴定神经元。结果：该方法培养的海马神经元生长状态良好，纯度较高。结论：采用低浓度胰酶消化，差速贴壁及无血清培养啮齿类海马神经元符合体外细胞实验要求，为进一步研究提供良好的目的细胞。

关键词：海马神经元；低浓度胰酶；差速贴壁；无血清培养

中图分类号 Q42 文献标识码 A 文章编号 1007-7731（2016）02-06-03

Culturing and Identification of the Neonatal Rodent Hippocampal Neurons

Chen Xuezhou1 et al.

（1 The Department of Orthopedics， the First Affiliated Hospital of Anhui Medical University， Hefei 230022，China）

Abstract：Obejctive：To established a simple， effective and stable method for the culture of rat hippocampal neurons in vitro， and provide target cell for dissecting molecular and cellular mechanisms in neuroscience.Methods：The hippocampus of neonatal rat were dissected and cell suspension were digested by low concentration of trypsin.The cell suspensions were cultured with serum-free Neurobasal medium after four hours differential adherence.Morphological observation of neurons growth state by inverted microscope， the hippocampal neurons can be identified by MAP2 specific antibody with DAPI.Results：The hippocampal neurons grew well and reached a high purity.Conclusion：Using low concentration of trypsin， differantial adherence and serum-free medium conforms to the requirements of neonatal rat hippocampal neurons culture in vitro， so as to provide targeted cells for further research.

Key words：Hippocampal neurons；Low concentration of trypsin；Differantial adherence；Serum-free medium

脊髓损伤（spinal cord injury，SCI）是脊柱外科一种致残率很高的疾病，易造成患者运动功能障碍，大小便失禁甚至终身瘫痪[1]。现代研究表明，脊髓损伤后，经历第一次原发性损伤及第二次继发性损伤，二次损伤是继发性，是可以预防和治疗的，其重点在于保护残存神经元的功能[2]。建立简单、稳定、高效的神经元体外培养是在细胞水平研究脊髓损伤的重要物质基础。

1 材料与方法

1.1 材料

1.1.1 动物 出生24h内SD乳鼠，雌雄不限，由安徽医科大学实验动物中心提供。

1.1.2 主要试剂 Neurobasal培养基、B27无血清添加剂、GlutaMAX-I添加剂、DMEM（high glucose）培养基、灭活胎牛血清、0.25%胰酶、台盼蓝染液均购自Invitrogen公司，多聚-D-赖氨酸、DPBS、Hanks液、抗荧……