简便_高效体外培养鼠小脑颗粒神经元的研究.pdfVIP

中国医科大学学报 第39 卷 第8 期 2010 年8 月 621 Journal of China Medical University Vol.39 No.8 Aug. 2010 · · 简便、高效体外培养小鼠小脑颗粒神经元的研究 谭斐，赵冬雪，李维帅，郭阳 （中国医科大学附属盛京医院神经内科，沈阳110004） 摘要：目的 简便、高效地分离和提纯小鼠小脑颗粒神经元，提高培养该神经元的纯度和活性。方法 通过采用简便、可行的细 胞分离方法，在出生7 d 的小鼠小脑组织中分离原代神经元。计数分离的原代神经元并分析其活性，免疫荧光染色证实其纯度， 观察其胞体和轴突生长情况。结果 分离的原代细胞活性可高达95% 以上；免疫荧光染色证实神经元占分离细胞的97% ；培 养24 h 后的神经元易于观察细胞突起的变化，培养48 h 后的神经元易于观察轴突的变化。结论 该方法方便、可行、高效，可 用以提纯小脑颗粒神经元进行体外神经元的生长发育、轴突再生和神经系统疾病的研究。 关键词：原代神经元培养；小脑颗粒神经元 中图分类号：R338.2 文献标志码：A 文章编号：0258-4646 （2010）08-0621-04 A Study to Simply and Efficiently Culture the Mouse Cerebellar Granule Neurons In Vitro TAN Fei ，ZHAO Dong-xue ，LI Wei-shuai，GUO Yang （Department of Neurology，Shengjing Hospital，China Medical University，Shenyang 110004，China） Abstract ：Objective To find and test an assay that can be used to get cerebellar granule neurons from mouse with high viability and high purity. Methods Cerebellar granule neurons were separated from 7-day old mouse. The cell viability was counted by dyeing the cells with trypan blue ，the cell purity was calculated by immunofluorescence staining with βⅢ-tubulin and GFAP，and the morphology of these cells were observed under microscope. Results By using the methods described in this study，we separated cerebellar granule neurons from 7- day old mouse，the viability of the cerebellar granule neurons was as high as 95% ；double staining with βⅢ-tubulin and GFPA showed that the purity of cerebellar granule neurons could reach 97%. Under microscope，after 24 or 48 hours of in vitro culture，neurite extention or the axons of the cerebellar granule neurons were easy to recognize. Conclusion The cerebellar granule neurons with high viability and high purity extracted by this method can be used for the studies of neuron outgrowth，neurogene