中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (32): 5186-5190.doi: 10.3969/j.issn.2095-4344.0383

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

一种高纯度原代神经元培养和高效率转染的方法

李兴统1，汤红艳3，马  微1，杨金伟1，2，王先斌1，代云飞1，李俊彦4，郭建辉2，李力燕1   

  1. 1昆明医科大学神经科学研究所，云南省昆明市  650500；2云南省第一人民医院普外二科，云南省昆明市  650032；3昆明医科大学第一附属医院神经外科，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
  • 收稿日期:2018-04-21 出版日期:2018-11-18 发布日期:2018-11-18
  • 通讯作者: 李力燕，博士，教授，昆明医科大学神经科学研究所，云南省昆明市 650500 并列通讯作者：郭建辉，主任医师，云南省第一人民医院普外二科，云南省昆明市 650032 并列通讯作者：李俊彦，副主任医师，昆明市第一人民医院神经外科，云南省昆明市 650032
  • 作者简介:李兴统，男，1991年生，云南省宣威市人，汉族，2017年昆明医科大学毕业，硕士，主要从事神经生物学研究。 并列第一作者：汤红艳，女，1969年生，云南省宜良县人，汉族，2010年北京大学医学网络教育学院毕业，主管护师，主要从事神经生物学研究。
  • 基金资助:

    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)

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

Li Xing-tong1, Tang Hong-yan3, Ma Wei1, Yang Jin-wei1, 2, Wang Xian-bin1, Dai Yun-fei1, Li Jun-yan4,
Guo Jian-hui2, Li Li-yan1   

  1. 1Institute for Neuroscience, Kunming Medical University, Kunming 650500, Yunnan Province, China; 2Second Department of General Surgery, the First People’s Hospital of Yunnan Province, Kunming 650032, Yunnan Province, China; 3Department of Neurosurgery, the First Affiliated Hospital of Kunming Medical University, Kunming 650032, Yunnan Province, China; 4Department of Neurosurgery, the First Hospital of Kunming, Kunming 650032, Yunnan Province, China
  • Received:2018-04-21 Online:2018-11-18 Published:2018-11-18
  • Contact: Li Li-yan, MD, Professor, Institute for Neuroscience, Kunming Medical University, Kunming 650500, Yunnan Province, China Corresponding author: Guo Jian-hui, Chief physician, Second Department of General Surgery, the First People’s Hospital of Yunnan Province, Kunming 650032, Yunnan Province, China Corresponding author: Li Jun-yan, Associate chief physician, Departement of Neurosurgery, the First Hospital of Kunming, Kunming 650032, Yunnan Province, China
  • About author:Li Xing-tong, Master, Institute for Neuroscience, Kunming Medical University, Kunming 650500, Yunnan Province, China Tang Hong-yan, Nurse-in-charge, Department of Neurosurgery, the First Affiliated Hospital of Kunming Medical University, Kunming 650032, Yunnan Province, China Li Xing-tong and Tang Hong-yan contributed equally to this work.
  • Supported by:

    the National Natural Science Foundation of China, No. 31560295 and 31260253; the Applied Basic Research Project of Yunnan Province, No. 2015FB098; the Health, Science and Technology Project of Yunnan Province, No. 2014NS202

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摘要：

文章快速阅读：

文题释义：
神经元原代培养：从动物的胚胎或新生动物的脑组织取下某一局部区域，分离细胞，培养在培养容器后不再移植，称为原代神经元培养。
人工脂质体转染：是一种人工膜，利用脂质体可以和细胞膜融合的特点，可用作外源物质进入细胞的载体，通过细胞内吞作用将DNA转移进入细胞，形成内含体，DNA从内含体释放，进入细胞质中，再进一步进入核内转录、表达。
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5×109 L-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。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程
ORCID: 0000-0002-0106-0844(李兴统)

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

Abstract:

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.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Neurons, Cell Culture Techniques, Transfection, Liposomes, Tissue Engineering

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