一种高纯度原代神经元培养和高效率转染的方法  

A method of high purity primary neuron culture and high efficiency transfection

作　　者：李兴统 汤红艳[2] 马微[1] 杨金伟[1,3] 王先斌 代云飞 李俊彦[4] 郭建辉[3] 李力燕[1] Li Xing-tong;Tang Hong-yan;Ma Wei;Yang Jin-wei;Wang Xian-bin;Dai Yun-fei;Li Jun-yan;Guo Jian-hui;Li Li-yan(Institute for Neuroscience,Kunming Medical University,Kunming 650500,Yunnan Province,China;Second Department of General Surgery,the First People’s Hospital of Yunnan Province,Kunming 650032,Yunnan Province,China;Department of Neurosurgery,the First Affiliated Hospital of Kunming Medical University,Kunming 650032,Yunnan Province,China;Department of Neurosurgery,the First Hospital of Kunming,Kunming 650032,Yunnan Province,China)

机构地区：[1]昆明医科大学神经科学研究所,云南省昆明市650500 [2]昆明医科大学第一附属医院神经外科,云南省昆明市650032 [3]云南省第一人民医院普外二科,云南省昆明市650032 [4]昆明市第一人民医院神经外科,云南省昆明市650032

出　　处：《中国组织工程研究》2018年第32期5186-5190,共5页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(31560295,31260253);云南省应用基础研究(昆医联合专项)(2015FB098);云南省卫生科技计划项目(2014NS202)。

摘　　要：背景:神经元的培养和细胞转染技术是在体外研究神经元生长发育及生理病理机制的重要手段,但目前原代培养的皮质神经元往往纯度不高、转染效率不高。目的:建立一种简单、高效、稳定的原代神经元培养及转染方法。方法:无菌条件下分离新生1 d SD大鼠大脑皮质,经0.25%的胰蛋白酶消化后离心,制备成单细胞悬液,以5×10~9L^(-1)细胞浓度接种至含神经元专用培养基的24孔细胞培养板进行原代培养,并于倒置显微镜下观察神经元形态的变化,使用神经元特异性标志物Tuj1和MAP2对培养的细胞进行鉴定;利用人工脂质体转染神经元,观察神经元转染效率。结果与结论:(1)体外培养的原代神经元具有较高的纯度,在培养第7天神经元特征明显,突起相互接触形成神经元网络,并表达神经元特异性标志物Tuj1与MAP2;利用人工脂质体转染神经元,转染效率较高;(2)采用该方法进行原代神经元培养,能够获得稳定的、高纯度的原代神经元;人工脂质体亦可高效率转染原代神经元。BACKGROUND:Neuron culture and cell transfection technologies are important means to study the development and pathophysiologic mechanism of neurons in vitro,but the purity and transfection efficiency of primary cultured cortical neurons are poor.OBJECTIVE:To establish a simple,efficient and stable method of culture and transfection method of primary neurons in vitro.METHODS:Primary cortical neurons were harvested from neonatal 1-day rat brains under aseptic condition,which was digested with 0.25%trypsin prior to centrifugation and made into cell suspensions,followed by being seeded into Neurobasal-A medium(5×109 L-1 per pore).The morphological characteristics of neurons were observed by inverted microscope;two neuron-specific markers(MAP2 and Tuj1)were used for immunolabeling to identify the cultured cells;the transfection efficiency of neurons was tested by Lipofectamine 2000 and Block-iT Transfection Kit.RESULTS AND CONCLUSION:The neurons cultured in vitro exhibited interconnected networks after culture for 7 days.All the cultured neurons displayed MAP2 and Tuj1 immunoreactivity.The highly effective transfection was observed under fluorescence microscope using Lipofectamine 2000 transfected neurons.In summary,the culture method of primary cerebral cortex neurons can be adopted to obtain highly purified neurons.Besides,high transfection efficiency of primary neurons can be realized by artificial liposome.

关 键 词：神经元 原代神经元培养 形态学鉴定 免疫荧光鉴定 转染 人工脂质体 纯度 组织构建 神经元 细胞培养技术 转染 脂质体 组织工程 

分 类 号：R318[医药卫生—生物医学工程]