SD大鼠乳鼠皮层神经元细胞原代培养模型的建立及鉴定  

Primary Culture and Identification of Cortical Neurons from Neonatal SD Rats

作　　者：李晓晓[1] 陈欣月 刘丽娜 李慧[2] 徐志卿[1,4] LI Xiao-xiao;CHEN Xin-yue;LIU Li-na;LI Hui;XU Zhi-qing(Department of Neurobiology,Beijing Key Laboratory of Neural Regeneration and Repair,Capital Medical University,Beijing,100069,China;Department of Anatomy,Capital Medical University,Beijing,100069,China;Central Laboratory,Capital Medical University,Beijing,100069,China;Department of Pathology,Capital Medical University,Beijing,100069,China)

机构地区：[1]首都医科大学基础医学院神经生物学系北京神经修复与再生重点实验室,北京100069 [2]首都医科大学基础医学院人体解剖与组织胚胎学系,北京100069 [3]首都医科大学中心实验室,北京100069 [4]首都医科大学基础医学院病理学系,北京100069

出　　处：《现代生物医学进展》2021年第21期4001-4005,4050,共6页Progress in Modern Biomedicine

基　　金：国家自然科学基金面上项目(81671345);北京市科技计划项目(Z181100001518001)。

摘　　要：目的:探讨SD大鼠乳鼠皮层神经元细胞原代培养方法,并鉴定其培养效果,以期建立一种生物学功能良好的体外细胞实验模型。方法:取出生24 h的SD大鼠乳鼠,分离出大脑皮层,在胰酶消化之前先进行离心,然后将胰酶消化后多次离心得到的细胞悬液接种于L-多聚赖氨酸包被的培养皿和共聚焦皿中,以加B27的Neurobasal-A培养基进行神经元细胞的原代培养,倒置显微镜下观察培养细胞的生长状态;通过免疫荧光组化的方法采用神经元标记物MAP-2进行神经元纯度的鉴定;在导入Fluo4-AM的原代神经元细胞,观察电刺激后胞内钙离子信号的变化,以验证神经元细胞的生理状态。结果:采用此方法培养的神经元细胞紧密贴壁、分散均匀、状态良好,神经元细胞周围突起相互连接形成网络;经MAP-2免疫荧光组化技术鉴定神经元的纯度达到95%以上;胞内钙离子信号的变化提示所培养的神经元具有良好的生物学功能。结论:该方法能获得纯度较高并且生物学功能良好的原代培养的SD大鼠乳鼠皮层神经元细胞。Objective:To investigate the method for culturing cortical neurons from neonatal SD rats.Methods:The cerebral cortex of 24-hour-old SD rats were isolated and centrifuged before trypsin digestion.The cell suspension obtained after trypsin digestion was planted into L-polylysine coated culture dish or confocal dish.The primary culture of neurons was carried out in a culture medium contained B27 and Neurobasal-A.The growth of cultured cells was checked under inverted microscope.Immunofluorescence histochemistry was used to identify the purity of neurons by using microtubule-associated protein 2(MAP-2)as a marker of neurons.The changes of intracellular calcium signal after electrical stimulation was observed in Fluo4-AM loaded primary neurons to verify the physiological state of neurons.Results:The neurons cultured with this method were closely adherent,evenly dispersed and in good condition.The neurites of the neurons were interconnected to form a network.MAP-2 immunofluorescence histochemistry showed that the purity of neurons was more than 95%.The stimulation-induced intracellular calcium signal indicated that the cultured neurons had good biological function.Conclusions:This method can make primary cultured cortical neurons of SD rats with high purity and good biological function.

关 键 词：SD大鼠乳鼠 皮层神经元 原代培养 纯度 钙离子信号 

分 类 号：R-33[医药卫生] Q593.2[生物学—生物化学]