中国组织工程研究

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (51): 7672-7677.doi: 10.3969/j.issn.2095-4344.2016.51.011

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

一种新生SD大鼠皮质源性神经元的培养方法

王东艳1，杨金伟2，程敬茹3，马  微1，李兴统1，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昆明理工大学医学院，云南省昆明市  650500
  • 收稿日期:2016-09-17 出版日期:2016-12-09 发布日期:2016-12-09
  • 通讯作者: 通讯作者： 李力燕，博士，教授，博士生导师，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，教授，硕士生导师，云南省第一人民医院普外二科，云南省昆明市 650032
  • 作者简介:王东艳，女，1990年生，河南省商丘市人，汉族，昆明医科大学在读硕士，主要从事神经生物学研究。
  • 基金资助:

    国家自然科学基金(31560295，31260253，81260075)；云南省应用基础研究昆医联合专项(2015FB098，2014FZ066)；云南省教育厅科学研究基金项目(2014C020Y)；2014年省级临床重点专科建设项目-普外科(云南省第一人民医院)

A culture method for cortical neurons derived from neonatal Sprague-Dawley rats

Wang Dong-yan1, Yang Jin-wei2, Cheng Jing-ru3, Ma Wei1, Li Xing-tong1, 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; 3Medical Faculty of Kunming University of Science and Technology, Kunming 650500, Yunnan Province, China
  • Received:2016-09-17 Online:2016-12-09 Published:2016-12-09
  • Contact: Corresponding author: Li Li-yan, M.D., Professor, Doctoral supervisor, Institute for Neuroscience, Kunming Medical University, Kunming 650500, Yunnan Province, China Corresponding author: Guo Jian-hui, Chief physician, Professor, Master’s supervisor, Second Department of General Surgery, the First People’s Hospital of Yunnan Province, Kunming 650032, Yunnan Province, China
  • About author:Wang Dong-yan, Studying for master’s degree, Institute for Neuroscience, Kunming Medical University, Kunming 650500, Yunnan Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 31560295, 31260253, 81260075; Priority Union Foundation of Yunnan Provincial Science and Technology Department and Kunming Medical University, No. 2015FB098, 2014FZ066; the Science Foundations of Education Department of Yunnan Province, No. 2014C020Y; the Provincial Key Construction Project in 2014-Department of General Surgery (the First People’s Hospital of Yunnan Province)

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摘要：

文章快速阅读：

文题释义：
大脑皮质：是大脑的表层，由灰质构成，其厚度为1-4 mm，其下方大部分由白质构成。大脑皮质的神经元都是多极神经元，按其细胞的形态分为锥体细胞、颗粒细胞和梭形细胞三大类。大脑皮质是调节躯体运动或者说控制躯体运动的最高级中枢。
神经元：又称神经细胞，是构成神经系统结构和功能的基本单位。具有长突起，由细胞体和细胞突起构成。细胞体位于脑、脊髓和神经节中，细胞突起可延伸至全身各器官和组织中。神经元的核大而圆，位于细胞中央，染色质少，核仁明显。常见的形态为星形、锥体形、梨形和圆球形状等。
摘要
背景：神经元的体外原代培养在研究神经系统的发育、再生、信号转导机制、神经药理学以及基因表达方面具有极其重要的地位。
目的：建立一种操作更加简单且能够得到较高纯度新生SD大鼠皮质神经元原代培养的方法。
方法：取1 d新生SD大鼠皮质。传统方法实验组：取整个皮质；改良方法实验组：取SD大鼠表面2.0-3.0 mm处皮质。木瓜蛋白酶消化后离心，制备成单细胞悬液，以1×105/孔的浓度接种至含神经元培养液的24孔培养板进行原代培养。培养3 d时采用免疫细胞化学染色方法，使用神经元特异性标志物Tuj1与MAP-2双标记法鉴定所培养的细胞；采用倒置相差显微镜观察6，24，48，72 h及5，7 d细胞数和突起长度并记录。
结果与结论：①所培养的细胞可表达神经元特异性标志物Tuj1与MAP-2，因此所培养细胞为神经元，可用于之后实验；②实验组培养至第3天时神经元的纯度已达到峰值92%，而普通实验组神经元的纯度为51%；③2组细胞培养至6 h均已贴壁且长处小突起，培养至第3天时，细胞已初步形成神经网络，培养至5 d时神经网络密集；④研究所用方法简便能够稳定的培养出纯度较高的神经元，可以用于SD大鼠皮质源性神经元的相关实验研究。

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: 0000-0002-8961-5807(王东艳)

关键词: 组织构建, 组织工程, 皮质神经元, SD大鼠, 细胞培养, 国家自然科学基金

Abstract:

BACKGROUND: Primary culture in vitro of neurons plays an important role in the development, regeneration, signal transduction mechanisms, neuropharmacology and gene expressions of the nervous system.
OBJECTIVE: To establish a simple method for primary culture of high-purity cortical neurons in neonatal Sprague-Dawley rats.
METHODS: Cortical tissues were acquired from neonatal Sprague-Dawley rats born 1 day. In traditional experimental group, the whole cortex was removed; in improved experimental group, the cortical tissues, 2-3 mm thick on the brain surface were removed. Single cell suspensions were prepared after papain digestion and centrifugation and were then seeded onto 24-well culture plates containing neuron solutions for primary culture (1×105 per well). Cells were identified by neuronal specific markers MAP-2 and Tuj1 after 3-day culture. The number of neurons and neurite length were observed under inverted phase contrast microscope and recorded at 6, 24, 48 and 72 hours, 5 and 7 days of culture, resprctively.
RESULTS AND CONCLUSION: The cultured cells expressing MAP-2 and Tuj1 were neurons that could be used in the following experiments. The purity of neurons in the improved experimental group was 92% at 3 days, while only 51% in the traditional experimental group. Cells in both two groups had attached to the wall presenting with small processes at 6 hours, and a simple neural network formed at the 3rd day until dense neural networks could be found at the 5th day. To conclude, our culture method herein is simple and convenient, and can be used to produce neurons with high purity, which will be helpful for the experimental studies on cortical neurons from Sprague-Dawley rats.

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, Cells, Cultured, Tissue Engineering

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