啮齿类海马神经元的原代培养及方法改进

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.%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 hip-pocampus 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:Us-ing low concentration of trypsin,differantial adherence and serum-free medium conforms to the requirements of neo-natal rat hippocampal neurons culture in vitro,so as to provide targeted cells for further research.