探讨单细胞记录和神经细胞培养时神经元的分离方法.pdfVIP

( ) ( ) 5·16 · 　山西医科大学学报 J Shanxi Med Univ 2005 年 8 月 , 36 4 　 ( ) 文章编号 : 　1007 - 6611 2005 04 - 0516 - 03 　 探讨单细胞记录和神经细胞培养时神经元的分离方法 郑肇青 , 　张 　宇 , 　张 　策 　( 山西医科大学神经生物学研究室 , 　太原 　030001) 摘要 : 　目的　探讨针对不同实验目的皮层神经元的最佳分离方法。方法 　针对单细胞记录分别采用蛋白酶 E 分离法和胰 蛋白酶分离法分离神经元 ,针对神经细胞培养分别采用胰蛋白酶分离法和机械分离法 ,之后通过形态学、膜片钳和染色法等 方法分别比较上述分离方法对神经元活性的影响。结果 　经蛋白酶 E 或胰蛋白酶分离法得到的细胞在进行单细胞记录时虽 然细胞的死亡率没有区别 ,但形态学、膜片钳等实验均表明蛋白酶 E 分离法分离的神经元活性更好 ;在神经细胞培养中经形 态学、膜片钳和台盼蓝染色法和凝胶电泳等方法都表明胰蛋白酶分离法分离的神经元活性好 ,更适于神经细胞培养。结论 　 单细胞记录时蛋白酶 E 分离法更为合适 ,神经细胞培养时胰蛋白酶分离法更为合适。 关键词 : 　细胞分离 ; 　细胞培养 ; 　神经元 ; 　膜片钳术 中图分类号: 　R33 - 33 　　　文献标识码 : 　A To explore the suitable methods to isolate cortical neurons for single neuronal recording or neuronal culture ( ) ZHEN G Zhaoqing ,ZHAN G Yu ,ZHAN G Ce Dept of Neurobiology , S hanxi Medical U niversity , Taiyuan 030001 , China Abstract : 　Objective 　To explore the suitable methods for cortical neurons in different experiments including single neuronal record ing and neuronal culture. 　Methods 　Cortical neurons were isolated by pronase E or trypsin for single cell record ,or by trypsin or mechanical methods for neuronal culture ,and the activeness of neurons were examined by morphological observation , patchclamp techniques ,trypan blue dyeing and gel electrophoresis techniques. 　Results 　Neurons isolated by pronase E were suitable for single record as examined by morphological observation and patchclamp method ,even though the death rates of neurons were no difference ; neurons isolated by trypsin were suitable for culture as examined by morphological observation ,patchclamp techniques ,trypan blue method and gel electrophoresis techniques. 　Conclusi