大脑皮层神经元原代培养方法的改良及一种体外缺氧缺糖简易模型的建立  

Improvement of primary cortical neuron culture and establishment of oxygen-glucose deprivation model

作　　者：周新华[1] 张在军[1] 于沛[1] 

机构地区：[1]暨南大学药学院新药研究所,广州510632

出　　处：《中南药学》2014年第5期439-442,共4页Central South Pharmacy

基　　金：国家重大新药创制-候选药物研究(No.2012ZX09103101-055);广东省医学科研基金项目(No.A2013353);暨南大学科研培育与创新基金项目(No.21611321和No.21613331)

摘　　要：目的改进SD胎鼠大脑皮层神经元体外原代培养方法及其一种缺氧缺糖(OGD)简易模型建立。方法用温和的TrypLE胰酶消化得到单个皮层神经细胞,用含有B27的Neurobasol培养基进行培养。培养7 d后,将其培养液换成无糖DMEM培养液,放入缺氧装置中分别培养4、5及6 h,利用MTT法检测细胞存活率,并用乳酸脱氢酶(LDH)试剂盒检测细胞培养液中LDH的释放量。结果分离和培养的神经元生长状态良好,免疫荧光方法证明该方法培养的大脑皮层神经元纯度可达到(93.2±1.3)%,能够达到实验研究的要求。细胞OGD 4、5及6 h后,细胞存活率分别为(52.7±1.3)%、(26.5±3.3)%及(20.8±1.1)%;LDH的释放量分别为(52.9±4.8)%、(89.3±1.3)%及(93.2±1.1)%。结论改良后的培养方法能够稳定地获得高纯度的SD胎鼠大脑皮层神经元,用厌氧发生装置替代三气培养箱建立了一种OGD简易模型,同时选择OGD 4 h作为缺氧缺糖模型最佳时间点。Objective To establish a stable and efficient primary culture method for cortical neurons obtained from pregnant Sprague-Dawley rats and oxygen-glucose deprivation （OGD） model. Methods Cerebral cortex isolated from E17-18 day SD rats was dissociated to single cells by TrypLE express enzyme, and was proceeded to culture medium containing neurobasal supplement with B27. The morphology of cortical neurons was observed during growth process, and immunofluorescence was applied to identify the cortical neurons. After 7 days, cell viability was assessed by MTT assay while cytotoxicity determined by lactate dehydrogenase （LDH） immediately after OGD. Results Neurons grew well with distinct outline and bright halation. Immunoftuorescence showed that the cultured cells were cortical neurons with high purity （93.2±1.3）%. Cortical neurons were exposed to OGD condition for 4, 5 and 6 h, and the cell viability was significantly reduced in a time-dependent manner at （52.7 ±1.3）%, （26.5±3.3）% and （20.8±1.1）%, respectively. However, LDH release level increased time-dependently at （52.9±4.8）%, （89.3 ±1.3）% and （93.2 ± 1.1）%, respectively. Conclusion The method is simple, reliable and practical for culturing primary cortical neurons with high purity. The cortical neurons exposed 4 h to OGD is choosen as the best condition for OGD model using Gas Pack Pouch bag.

关 键 词：SD胎鼠 神经元 原代培养 TrypLE OGD MTT LDH 

分 类 号：R965.1[医药卫生—药理学]