新生大鼠海马神经元的分离培养及鉴定  

Isolation, culture and identification of hippocampal neurons from newborn rats

作　　者：金正旭 丁韵涵 王小娟 李小丽[1] 尹跃霏 张德奎[1] Jin Zheng-xu;Ding Yun-han;Wang Xiao-juan;Li Xiao-lil;Yin Yue-fei;Zhang De-kui(Department of Gastroenterology,The Second Hospital of Lanzhou University,Lanzhou 730030,China;Institute of Genetics,School of Basic Medical Sciences,Lanzhou University,Lanzhou 730000,China)

机构地区：[1]兰州大学第二医院消化内科,甘肃兰州730030 [2]兰州大学基础医学院遗传研究所,甘肃兰州730000

出　　处：《兰州大学学报（医学版）》2020年第3期20-22,27,共4页Journal of Lanzhou University（Medical Sciences）

基　　金：国家自然科学基金资助项目(81770525);甘肃省青年基金资助项目(18JR3RA326,18JR3RA332)。

摘　　要：目的探讨新生大鼠原代海马神经元的分离培养及鉴定方法。方法选取新生24 h内SD大鼠,处死后分离海马体,运用机械分离及胰蛋白酶消化法制备单细胞悬液,离心后以5.0×10^5/mL的密度种植于24孔板内,每3~4 d更换培养液的1 2~1 3。结果提取的原代海马神经元于24 h后贴壁,并逐渐生长为梭形;培养3 d后神经元形成突起,逐渐增多,并不断延伸;培养4 d后开始形成清晰的神经纤维网络,胞体丰满,胞体周围可见光晕,贴壁良好;培养10 d后神经元发育更加成熟,神经纤维网络更加密集。荧光显微镜下,可见微管相关蛋白在海马神经元细胞体和树突中表达,神经元细胞阳性率为95%以上。结论经改良既往实验方法,得到可培养出细胞活性高、神经元阳性率高的新生大鼠海马神经元体外培养方法。Objective To explore the methods of isolation and culture and identification of primary hippocampal neurons of neonatal rats. Methods Newborn SD rats were selected and their hippocampi were isolated after sacrifice. Single cell suspensions were prepared by mechanical separation and trypsin digestion. After centrifugation, they were planted in 24-well plates at a density of 5.0×10^5/mL. Change the 1/2~1/3 of the culture solution every 3~4 days. Results The extracted primary hippocampal neurons adhered to the wall 24 hours later and gradually grew into a fusiform shape. After 3 days of culture, the neurons formed protrusions, gradually increased and continued to extend. After 4 days, a clear neural fiber network was formed, the neuron cell body was formed. The cell body of the neuron was full, with visible light around the cell body, adhering well.After 10 days of culture, the neurons developed more maturely and the neural fiber network was more dense.Under the fluorescence microscope, microtubule-associated protein was expressed in hippocampal neurons and dendrites, the positive rate of neuronal cells was over 95%. Conclusion The previous experimental methods and obtained a method for in vitro culture of neonatal rat hippocampal neurons with high cell viability and high neuron positive rate.

关 键 词：海马神经元 细胞培养 微管相关蛋白2抗体 

分 类 号：R331[医药卫生—人体生理学]