木瓜蛋白酶联合DNA酶法提取乳鼠原代皮层神经元及鉴定  

Extraction and identification of primary cortical neurons of suckling rats by papain and DNA enzyme

作　　者：廖益东 明江 张宇 廖一飞 徐卡娅[1,2] LIAO Yidong;MING Jiang;ZHANG Yu;LIAO Yifei;XU Kaya(Guizhou Medical University,Guiyang 550004,China;Affiliated Hospital of Guizhou Medical University)

机构地区：[1]贵州医科大学,550004 [2]贵州医科大学附属医院

出　　处：《天津医药》2022年第6期583-587,共5页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(81901173,82060231);贵州省普通高等学校青年科技人才成长项目(黔教合KY字[2021]190)。

摘　　要：目的采用木瓜蛋白酶联合DNA酶法提取24 h内新生SD大鼠皮层神经元,改进体外原代培养方法。方法取24 h内新生SD大鼠,剥离脑血管膜分离出大脑皮层,采用木瓜蛋白酶和DNA酶顺序消化、纯化后,以含10%胎牛血清的DMEM-F12培养基为神经元接种液,4 h后更换为预热的含有B27的Neurobasal-A神经元培养液,连续培养7 d。分别以1×10^(5)、5×10^(5)、1×10^(6)细胞/mL接种于L-多聚赖氨酸浸泡后的6孔板内,并在显微镜下观察细胞的生长状态。取培养7 d后的神经元,采用β-Tubulin免疫荧光法和神经元特异性核蛋白(Neun)抗体免疫组化法鉴定神经元,神经元微管相关蛋白2抗体(MAP2)免疫荧光法鉴定神经元纯度。结果神经元以密度为5×10^(5)细胞/mL接种最为合适,接种24 h后,皮层神经元部分贴壁;接种3 d后,贴壁细胞逐渐增多,神经元突触进一步生长并延长,交联成稀疏网络;接种7 d后,神经元仍大量生长,胞体丰满,并形成更为紧密的神经元网络系统。经β-Tubulin免疫荧光法和Neun抗体免疫组化法鉴定,提取培养细胞为神经元,并经神经元标志物MAP2免疫荧光法鉴定神经元纯度为(91.06±1.51)%。结论采用24 h内新生SD大鼠分离大脑皮层,经木瓜酶与DNA酶顺序消化,可提取到优质皮层神经元。Objective enzyme,and to improve the primary culture method in vitro.Methods separated from the cerebral vascular membrane within 24 h.After sequentially digested and purified by papain and DNA enzyme,the neurons were inoculated with DMEM-F12 medium containing 10%fetal bovine serum.After 4 hours,it was replaced with pre-warmed Neurobasal-A neuronal medium containing B27,and cultured continuously for 7 days.1×10^(5),5×10^(5)and 1×10^(6)cells/mL were seeded into 6-well plates soaked in L-polylysine,respectively and the growth state of the cells was observed under a microscope.The neurons cultured for 7 days were identified by immunofluorescence method ofβ-Tubulin and immunohistochemical method of neuron specific nuclear protein(Neun)antibody.The purity of neurons was identified by immunofluorescence method of neuron microtubule-associated protein 2 antibody(MAP2).Results The most suitable inoculation method was 5×10^(5)cells/mL/well.The cortical neurons partially adhered to the wall after 24-h inoculation.Three days after inoculation,adherent cells gradually increased,synapses further grew and extended,and crosslinked into sparse networks.Seven days after inoculation,the neurons still grew in large numbers,the cell body was plump,and a tighter neural network system was formed.The cells were identified by immunofluorescence method ofβ-Tubulin and immunohistochemical method of Neun antibody,and the neuron purity was(91.06±1.51)%by immunofluorescence method of neuron marker MAP2.Conclusion Newborn SD rats within 24 hours are used to separate the cerebral cortex,and after digestion with papain and DNA enzyme,high-quality cortical neurons can be extracted.

关 键 词：神经元 原代细胞培养 木瓜蛋白酶 脱氧核糖核酸酶类 大鼠 Sprague-Dawley 新生SD大鼠 细胞模型 

分 类 号：R329-33[医药卫生—人体解剖和组织胚胎学]