一种改进的小脑颗粒神经元原代培养方法及其性质鉴定  

An improved primary culture method of cerebellar granule neurons and its characterization

作　　者：陈海云 CHEN Hai-yun(School of Pharmacy, Guangdong Pharmaceutical University in Guangzhou, Guangzhou 510006, China)

机构地区：[1]广东药科大学药学院药理系,广州广东510006

出　　处：《中国药理学通报》2020年第10期1476-1480,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金青年基金(No 81803512);广东省自然科学基金博士科研启动项目(No 2018A030310562)。

摘　　要：目的在已报导的原代小脑颗粒细胞(cerebellar granule neurons,CGNs)的提取培养方法基础上进行改进,以获得较少杂质、更高存活率及更高纯度的CGNs,为后续体外研究做准备。方法将Tryple Express消化液消化及前后两次离心后得到的单细胞悬液,经台盼蓝活细胞计数后接种在预先用多聚赖氨酸(poly-L-lysine,PLL)包被的培养板中。接种20~24 h后,加入10μmol·L-1的阿糖胞苷(cytosine arabinoside,Ara-C),以及从d 4开始每隔3天加入终浓度5 mmol·L-1的Glucose。借助荧光显微镜及神经元特异性标志物微管蛋白(β-Ⅲ-tubulin)对CGNs细胞进行形态观察及纯度鉴定。结果与原有的胰酶提取方法相比,改进后的CGNs提取方法细胞生长状态良好、突触发育完善、细胞碎片等杂质含量较少,并具有较高存活率及纯度(97%)。结论该方法简便可行,结果稳定,可以为体外神经元的生长发育、轴突再生和神经系统疾病的研究提供有力支持。Aim To obtain more neurons with higher purity and longer growth time in vitro using a new primary culture method of cerebellar granule neurons(CGNs).Methods After pre-coated with poly-L-lysine(PLL),CGNs isolated from 7-8 days old Sprague-Dawley rats were digested into single cells by Tryple Express enzyme,and were preceded to culture basal modified Eagle’s medium supplement with glutamax.After CGNs seeded in plates at the density of 1.5×106/mL for 20-24 h,cytosine arabinoside(Ara-C,10μM)was added to inhibit the growth of non-neuronal cells,and glucose(5 mmol·L^-1)was supplemented into the medium for cell energy metabolism and water evaporation.The cell morphology was observed by inverted phase contrast microscope,and the purity of CGNs cells was identified byβ-Ⅲ-tubulin.Results Compared with the original method of trypsin chemical digestion to separate cells,the yield of the improved method increased significantly since the Tryple Express enzyme was gentle with minor injury to the cells.After inoculation,the CGNs were evenly dispersed,with less cell debris and clean background.The dendrites and synapses of CGNs grew well with long survival.Finally,the purity of the improved method was over 97%confirmed by immunofluorescence staining.Conclusions The improved method is simple,replicable and stable,which could provide strong support for the study of neuron growth and development,axon regeneration and nervous system diseases in vitro.

关 键 词：小脑颗粒神经元 Tryple Express 阿糖胞苷 提取 β-Ⅲ-tubulin 免疫荧光染色 

分 类 号：R-332[医药卫生] R322.81