一种新生SD大鼠皮质源性神经元的培养方法

发布时间：2018-04-17 08:10

本文选题：神经元 + 细胞　；参考：《中国组织工程研究》2016年51期

【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。
[Abstract]:Background: primary culture of neurons in vitro plays an important role in studying the development, regeneration, signal transduction mechanism, neuropharmacology and gene expression of nervous system.Aim: to establish a simple method for primary culture of cortical neurons in neonatal SD rats.Methods: the cortex of 1 day newborn SD rats was taken.The whole cortex was taken from the experimental group and the cortex from 2.0 mm to 3.0 mm on the surface of SD rats.After digesting papain, a single cell suspension was prepared and inoculated into a 24 well culture plate containing neuron culture medium at a concentration of 1 脳 10 5 / well for primary culture.The cultured cells were identified by immunocytochemical staining with neuron specific markers Tuj1 and MAP-2 at 3 days, and the number of cells and the length of processes were observed and recorded by inverted phase contrast microscope.Results and conclusion the cells cultured at 1: 1 could express the neuron-specific markers Tuj1 and MAP-2.Therefore, the cultured cells were neurons.The purity of neurons in the experimental group reached the peak value at the 3rd day, while the purity of the neurons in the ordinary experimental group was 51kW. The cells in the experimental group were all adherent to the wall and had small protrusions until 6 h, and at the third day of culture, the purity of the neurons in the experimental group reached the peak, and the purity of the neurons in the experimental group reached the peak at the third day.The neural network has been formed preliminarily, and the method of dense neural network is simple and stable to produce high-purity neurons, which can be used to study cortical neurons in SD rats.
【作者单位】：昆明医科大学神经科学研究所;云南省第一人民医院普外二科;昆明理工大学医学院;
【基金】：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)~~
【分类号】：R338

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