中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (15): 2344-2349.doi: 10.12307/2023.390

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

新生SD大鼠大脑皮质神经元的原代培养及鉴定

王  京，卢思彤，吴印林，刘玉东，邹维艳，孙美群   

  1. 蚌埠医学院组胚教研室，安徽省蚌埠市   233030
  • 收稿日期:2022-03-08 接受日期:2022-06-27 出版日期:2023-05-28 发布日期:2022-10-17
  • 通讯作者: 孙美群，教授，硕士生导师，蚌埠医学院组胚教研室，安徽省蚌埠市 233030
  • 作者简介:王京，女，1996年生，安徽省芜湖市人，汉族，在读硕士，主要从事癫痫发病机制研究。 卢思彤，女，1995年生，安徽省宿州市人，汉族，在读硕士，主要从事癫痫发病机制研究。
  • 基金资助:
    安徽省高等学校自然科学研究重大项目(KJ2019ZD26)，项目负责人：孙美群；慢性疾病免疫学基础与临床安徽省重点实验室开放课题(KLICD-2022-Z4) ，项目负责人：孙美群

Primary culture and identification of neurons from cerebral cortex of newborn Sprague-Dawley rats

Wang Jing, Lu Sitong, Wu Yinlin, Liu Yudong, Zou Weiyan, Sun Meiqun   

  1. Department of Histology and Embryology, Bengbu Medical College, Bengbu 233030, Anhui Province, China
  • Received:2022-03-08 Accepted:2022-06-27 Online:2023-05-28 Published:2022-10-17
  • Contact: Sun Meiqun, Professor, Master’s supervisor, Department of Histology and Embryology, Bengbu Medical College, Bengbu 233030, Anhui Province, China
  • About author:Wang Jing, Master candidate, Department of Histology and Embryology, Bengbu Medical College, Bengbu 233030, Anhui Province, China Lu Sitong, Master candidate, Department of Histology and Embryology, Bengbu Medical College, Bengbu 233030, Anhui Province, China
  • Supported by:
    Natural Science Research Major Project of Anhui Provincial University, No. KJ2019ZD26 (to SMQ); Anhui Provincial Key Laboratory of Chronic Disease Immunology Basic and Clinical Open Project, No. KLICD-2022-Z4 (to SMQ)

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摘要：

文题释义：
微管相关蛋白：是细胞内存在的与微管结合的一种蛋白质，主要包括微管相关蛋白1、微管相关蛋白2、Tau和微管相关蛋白4等，前3种蛋白主要存在于神经元中，而微管相关蛋白4存在于各种细胞中。
胶质纤维酸性蛋白：是一种中间丝蛋白，分子质量为56 kD，主要存在于中枢神经系统的星形胶质细胞内，可作为一种特异性标志物用于鉴定星形胶质细胞。

背景：培养神经元的质量是研究中枢神经系统疾病的关键所在，但培养方法一直没有统一的标准。
目的：探究取材部位及阿糖胞苷处理对原代培养神经元活性、纯度及成熟度的影响。
方法：选取新生24 h内的SD乳鼠，取大脑皮质表面2.0-3.0 mm处细胞，培养后24，48，72 h，用5 μmol/L或10 μmol/L阿糖胞苷处理48 h；另取全部大脑皮质细胞，培养后48 h用5 μmol/L阿糖胞苷处理48 h。培养至第7天，倒置显微镜下观察细胞形态，采用CCK-8法检测阿糖胞苷对神经元活性的影响，免疫荧光和Western blot检测神经元的纯度及成熟度。
结果与结论：①倒置显微镜：10 μmol/L 阿糖胞苷24，48，72 h组神经元密度分别低于同时间下的5 μmol/L阿糖胞苷组，该6组神经元突起相互之间均可连接成紧密的网状结构，神经元胞体丰满并充满折光特性；全皮质组细胞密度低于正常对照组，并可明显观察到非神经元；②CCK-8检测：各阿糖胞苷组细胞活性均低于正常对照组(P < 0.001)， 10 μmol/L 阿糖胞苷24，48，72 h组神经元活性低于同时间下的5 μmol/L阿糖胞苷组(P < 0.05)，5 μmol/L 阿糖胞苷24 h组神经元活性低于 5 μmol/L 阿糖胞苷48，72 h组(P < 0.01)；③免疫荧光染色：各阿糖胞苷组神经元纯度大于正常对照组、全部皮质组(P < 0.001)，各阿糖胞苷组神经元纯度无差异；④Western blot检测神经元特异性烯醇化酶蛋白表达：各阿糖胞苷组神经元成熟度高于正常对照组(P < 0.05)，其中以 5 μmol/L 阿糖胞苷48 h组成熟度最高；⑤结果表明：取新生24 h内大鼠大脑皮质表面2.0-3.0 mm处皮质，培养后48 h加入5 μmol/L 阿糖胞苷处理得到的神经元活性、成熟度和纯度较好。
https://orcid.org/0000-0003-2938-2738(王京) 

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: 阿糖胞苷, 大脑皮质, 神经元, 原代培养, 神经元活性, 大鼠

Abstract: BACKGROUND: The quality of cultured neurons is the key to the study of central nervous system diseases, but there has been no unified standard for the methods used.

OBJECTIVE: To explore the effects of best selection site and cytarabine treatment conditions on the viability, purity and maturity of primary cultured neurons. 

METHODS:  The cells were obtained at 2.0-3.0 mm on the surface of the cerebral cortex of Sprague-Dawley rats within 24 hours of the newborn, and treated with cytarabine at a concentration of 5 μmol/L or 10 μmol/L at 24, 48, and 72 hours after culture. In addition, the whole cerebral cortex was removed for primary culture and treated with cytarabine at a concentration of 5 μmol/L for 48 hours at 48 hours after culture. At 7 days after culture, the morphology of neuron was observed using inverted microscope and the effect of cytarabine on neuronal viability was detected by CCK-8 assay. The purity and maturity of neurons were assessed by immunofluorescence and western blot assay. 

RESULTS AND CONCLUSION: (1) Inverted microscope: The density of neurons in the 10 μmol/L cytarabine group at 24, 48, and 72 hours was lower than that in the 5 μmol/L cytarabine group at the same time point, and the neuronal processes in the six groups could be connected to each other to form a dense network structure. The neuron cell body was plump and full of refractive properties. The cell density of the whole cortex group was lower than that of the normal control group, and non-neuronal cells could be clearly observed. (2) CCK-8 assay: The cell viability of each cytarabine group was lower than that of the normal control group (P < 0.001), and the neuronal viability of the 10 μmol/L cytarabine 24-, 48-, and 72-hour groups was lower than that of 5 μmol/L cytarabine group at the same time point (P < 0.05). The neuronal viability in the 5 μmol/L cytarabine 24-hour group was lower than that in the 5 μmol/L cytarabine 48- and 72-hour group (P < 0.01). (3) Immunofluorescence staining: The purity of neurons in each cytarabine group was greater than that in the normal control group and all cortical groups (P < 0.001), and there was no difference in the purity of neurons in each cytarabine group. (4) Western blot assay detection of neuron-specific enolase protein expression: The neuron maturity of each cytarabine group was higher than that of the normal control group (P < 0.05), and the 5 μmol/L cytarabine 48-hour group had the highest maturity. (5) The results showed that the cortex at 2.0-3.0 mm on the surface of the rat cerebral cortex within 24 hours of the newborn treated with 5 μmol/L cytarabine at 48 hours after culture can obtain better neuronal viability, maturity and purity.

Key words: cytarabine, cerebral cortex, neuron, primary culture, neuronal viability, rat

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