中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (6): 897-902.doi: 10.12307/2023.242

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

一种同时培养原代皮质及海马神经元的实验方法

廖益东1，明  江1 ，宋文学1，王梓力1，张  宇2，廖一飞2，徐卡娅2，杨  华2   

  1. 1贵州医科大学，贵州省贵阳市   550004；2贵州医科大学附属医院神经外科，贵州省贵阳市   550004
  • 收稿日期:2022-01-25 接受日期:2022-04-18 出版日期:2023-02-28 发布日期:2022-08-11
  • 通讯作者: 杨华，硕士，主任医师，贵州医科大学附属医院神经外科，贵州省贵阳市 550004
  • 作者简介:廖益东，男，1992年生，贵州省遵义市人，瑶族，贵州医科大学在读硕士，主要从事干细胞治疗脑血管疾病的作用机制的研究。
  • 基金资助:
    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)，项目负责人：徐卡娅；贵州医科大学2018年度学术新苗培养及创新探索专项项目(黔科合平台人才[2018]5779-44)，项目负责人：徐卡娅；国家自然科学基金项目(81901173，82060231)，项目负责人：徐卡娅

An experimental method for simultaneously culturing primary cortical and hippocampal neurons

Liao Yidong1, Ming Jiang1, Song Wenxue1, Wang Zili1, Zhang Yu2, Liao Yifei2, Xu Kaya2, Yang Hua2   

  1. 1Guizhou Medical University, Guiyang 550004, Guizhou Province, China; 2Department of Neurosurgery, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • Received:2022-01-25 Accepted:2022-04-18 Online:2023-02-28 Published:2022-08-11
  • Contact: Yang Hua, Master, Chief physician, Department of Neurosurgery, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • About author:Liao Yidong, Master candidate, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • Supported by:
    the Youth Science and Technology Talents Growth Project of General Colleges and Universities in Guizhou Province, No. KY [2021] 190 (to XKY); Special Project of Academic Seedling Cultivation and Innovation Exploration of Guizhou Medical University in 2018, No. [2018]5779-44 (to XKY); National Natural Science Foundation of China, No. 81901173, 82060231 (to XKY)

PDF

27

摘要：

文题释义：
原代神经元细胞：是中枢神经系统重要的组成部分，此种细胞可用作神经系统体外疾病模型，并广泛运用于神经细胞生物学特性和外源干扰因素的疾病研究中，其在神经生物学、发育生物学体外实验研究中扮演重要角色。
细胞疾病模型：是一种能在体外培养并能模拟人类疾病的新型技术，在疾病药理学、个体化治疗、病理生理发生机制及再生医学研究中均具有巨大的运用潜能，弥补了许多疾病无法用动物模拟且构建的动物模型存在个体差异等缺点，同时此项技术为人类疾病的研究奠定了基础。

背景：近年来原代神经元细胞模型在颅脑疾病中扮演着重要的角色，此类细胞模型特性更加贴近于疾病细胞，可用于模拟研究各类神经系统疾病。
目的：建立一种便捷实用的可同时提取皮质及海马原代神经元的培养方法。
方法：取新生24 h内的SD大鼠，麻醉后断脊法处死，采用体积分数为75%乙醇消毒，用镊子分离出颅骨及脑膜，解剖出完整大脑，剥离脑血管膜，游离出大脑皮质及海马组织，采用木瓜蛋白酶及适量DNA酶顺序消化方案，吹打、离心、过滤，然后接种于左旋多聚赖氨酸包被的6孔板和爬片内，以含体积分数10%胎牛血清的DMEM-F12培养基为种植液，6 h后换用含有B27的Neurobasal无血清专用培养基，持续培养7 d后，显微镜下观察细胞形态及生长状态。分别采用β-Tubulin免疫荧光法、Neun抗体免疫组化法鉴定皮质神经元与海马神经元，以及采用MAP2免疫荧光法鉴定其纯度。
结果与结论：①接种24 h后细胞体积变清晰，呈不规则圆形、周围环绕光晕，少数细胞已有细小突起，均已贴壁生长；持续培养3 d后，胞体逐步增大，部分呈聚团性生长，突触延长，细胞间出现交联；持续培养7 d后，胞体成熟饱满，细胞质显著增多，光晕增强，形成更为密集的神经元网络；②经Neun抗体免疫组化法、β-Tubulin免疫荧光法鉴定为神经元，神经元标志物MAP2免疫荧光法鉴定皮质神经元和海马神经元纯度分别为(94.00±0.34)%和(91.00±0.26)%，可用于后期实验；③结果表明，采用同一批24 h内新生SD大鼠分离大脑皮质和海马组织，经木瓜蛋白酶与DNA酶顺序消化后，可提取到优质的海马神经元及皮质神经元。该方案得到的神经元纯度高，操作简化，可作为研究各类神经系统疾病细胞模型的基础。

https://orcid.org/0000-0001-7324-248X (廖益东)

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 新生SD大鼠, 皮质神经元, 海马神经元, 原代培养, 神经疾病, 细胞模型

Abstract: BACKGROUND: In recent years, primary neuronal cell models have played an important role in brain diseases. The characteristics of such cell models are closer to disease cells and can be used to simulate various neurological diseases.  
OBJECTIVE: To establish a convenient and practical culture method that can simultaneously extract primary cortical and hippocampal neurons.
METHODS:  The SD rats within 24 hours of the newborn were sacrificed by chiropractic method, and then sterilized with 75% ethanol. After separating the skull and meninges using forceps, the whole brain was dissected out. The cerebrovascular membrane was stripped, and the cerebral cortex and hippocampus were dissociated. The sequential digestion protocol of papain and appropriate amount of DNase was used. After pipetting, centrifugation, and filtration, the samples were inoculated into L-polylysine-coated six-well plates and slides. DMEM-F12 medium containing 10% fetal bovine serum was used as the seeding medium. 6 hours later, the serum-free special medium containing Neurobasal B27 was used. After culturing for 7 days, the cell morphology and growth state were observed under the microscope. The cortical and hippocampal neurons were identified by β-Tubulin immunofluorescence method and Neun antibody immunohistochemistry. The marker MAP2 immunofluorescence method was applied to identify the purity.  
RESULTS AND CONCLUSION: (1) 24 hours after inoculation, the volume of cells became clear, presented irregular circles, surrounded by halos, and a few cells had small protrusions, all of which had grown adherently to the wall. After 3 days of continuous culture, the cell bodies gradually increased; some of them grew in clusters; synapses were elongated; and cross-links appeared between cells. After 7 days of continuous culture, the cell body was mature and full; the cytoplasm was significantly increased; the halo was enhanced; and a denser neuronal network was formed. (2) Neurons were identified by Neun antibody immunohistochemistry and β-Tubulin immunofluorescence method. The purities of cortical and hippocampal neurons were (94.00±0.34)% and (91.00±0.26)%, respectively detected using neuronal marker MAP2 immunofluorescence method. Neuronal cells could be used for experiments. (3) These results suggest that the cerebral cortex and hippocampus were isolated from the same batch of neonatal SD rats within 24 hours. After sequential digestion with papain and DNase, high-quality hippocampal and cortical neurons can be extracted. The neurons obtained by this protocol have high purity and simplified operation, and can be used as the basis for various neurological disease cell models.

Key words: neonatal SD rat, cortical neuron, hippocampal neuron, primary culture, neurological disease, cell model

中图分类号: