中国组织工程研究

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (12): 1915-1920.doi: 10.3969/j.issn.2095-4344.2017.12.019

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

阿糖胞苷在大鼠皮质神经元细胞培养中的适宜介入时间

关  宏1，潘学峰2，3，刘昊坤4，刘小青1，张丽娜2，王少毅2，董晓东2，牛嗣云2   

  1. 1保定市第一中心医院神经外科， 河北省保定市 071000；2河北大学医学院， 河北省保定市  071000；3北京理工大学生命学院，北京市  100081；4解放军军事交通学院第九队，天津市  300171
  • 收稿日期:2017-02-08 出版日期:2017-04-28 发布日期:2017-05-16
  • 通讯作者: 牛嗣云，博士，教授，河北大学医学院, 河北省保定市 071000
  • 作者简介:关宏，男，1977年生，河北省保定市人，满族，2011年河北大学医学院毕业，副主任医师，主要从事神经外科专业的研究。 并列第一作者：潘学峰，男，1966年生，河北省廊坊市人，汉族，1997年中国科学院毕业，博士，主要从事分子医学研究。
  • 基金资助:

    河北省自然科学基金(B2015201161)；国家自然科学基金(81502477)

Optimal acting time of cytarabine in primary culture of rat cortical neurons

Guan Hong1, Pan Xue-feng2, 3, Liu Hao-kun4, Liu Xiao-qing1, Zhang Li-na2, Wang Shao-yi2, Dong Xiao-dong2, Niu Si-yun2   

  1. 1Department of Neurosurgery, Baoding No. 1 Central Hospital, Baoding 071000, Hebei Province, China; 2School of Medicine, Hebei University, Baoding 071000, Hebei Province, China; 3School of Life Science, Beijing Institute of Technology, Beijing 100081, China; 4the 9th Team of Military Transportation University, Tianjin 300171, China
  • Received:2017-02-08 Online:2017-04-28 Published:2017-05-16
  • Contact: Niu Si-yun, M.D., Professor, School of Medicine, Hebei University, Baoding 071000, Hebei Province, China
  • About author:Guan Hong, Associate chief physician, Department of Neurosurgery, Baoding No. 1 Central Hospital, Baoding 071000, Hebei Province, China Pan Xue-feng, M.D., School of Medicine, Hebei University, Baoding 071000, Hebei Province, China; School of Life Science, Beijing Institute of Technology, Beijing 100081, China Guan Hong and Pan Xue-feng contributed equally to this work.
  • Supported by:

    the Natural Science Foundation of Hebei Province, No. B2015201161; the National Natural Science Foundation of China, No. 81502477

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摘要：

文章快速阅读：

文题释义：
阿糖胞苷：简写Ara-C，为主要作用于细胞S增殖期的嘧啶类抗代谢药物，通过抑制细胞DNA的合成，干扰细胞的增殖。本品为细胞周期特异性药物，对处于S期增殖期细胞的作用最敏感，对抑制RNA及蛋白质合成的作用较弱。阿糖胞苷分子式为C9H13N3O5，相对分子质量243.217。阿糖胞苷为白色粉末，可溶于水、乙醇和氯仿，在水溶液中尚稳定，水溶解度为0.4 g/mL。
神经元特异性烯醇化酶：是参与糖酵解途径的烯醇化酶中的一种，存在于神经组织和神经内分泌组织中。神经元特异性烯醇化酶在脑组织细胞的活性最高，外周神经和神经分泌组织的活性水平居中，最低值见于非神经组织、血清和脊髓液。它被发现在与神经内分泌组织起源有关的肿瘤中，特别是小细胞肺癌中有过量的神经元特异性烯醇化酶表达，导致血清中神经元特异性烯醇化酶明显升高。
摘要
背景：由于中枢神经系统疾病研究需求，实验室常用有毒性的阿糖胞苷获得纯度较高的神经元细胞，然而阿糖胞苷的介入时间鲜见研究。
目的：验证终浓度为10 μmol/L阿糖胞苷在大鼠皮质神经元原代细胞培养中的适宜介入时间。
方法：采用神经元专用培养基Neurobasal+B27进行大脑皮质组织原代神经元培养，分别于接种细胞后12， 24，36，48 h加入终浓度为10 μmol/L阿糖胞苷。每48 h半量换液。于7 d后倒置的显微镜下观察神经元形态；采用免疫细胞化学的方法对神经元特异性烯醇化酶进行免疫化学染色，鉴定神经元细胞的纯度和成熟度；计算机多功能图像分析系统对所有神经元特异性烯醇化酶阳性细胞进行形态学计量分析。
结果与结论：①接种细胞后24 h加入阿糖胞苷，神经元纯度在90%以上，分化成熟神经元细胞占比89.00%，神经元细胞胞体面积最大，突触最长，胞质透亮，核大而明显，细胞体周围折光性强，立体感强，突起三四个，具有三四级分叉，形成良好的网络结构，非神经元细胞较少，神经元纯度及细胞状态最佳；②结果表明，阿糖胞苷介入培养过程越早，神经元细胞纯度越高，但阿糖胞苷对神经元细胞分化成熟的影响也会越明显；采用neurolbasal培养基，于接种细胞后24 h加入阿糖胞苷，可以获得理想纯度，成熟度较好，生长状态较好的大脑皮质神经元。

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: 0000-0001-6861-3637(关宏)

关键词: 组织构建, 组织工程, 阿糖胞苷, 神经元培养, 神经元纯度, 国家自然科学基金

Abstract:

BACKGROUND: Toxic cytarabine is often used to prepare highly purified neurons in experimental studies addressing central nervous system diseases. However, the intervention time of cytarabine is little reported.
OBJECTIVE: To determine the optimal intervention time of cytarabine(final concentration 10 μmol/L) in primary culture of rat cortical neurons.
METHODS: Rat primary cortical neurons were cultured in Neurobasal+B27 medium, and 10 μmol/L cytarabine were added at 12, 24, 36 and 48 hours after culture, respectively. Half of the medium were changed every 48 hours. The morphology of neurons was observed under inverted microscope at 7 days. The purity and differentiation of neurons maturity were identified by immunocytochemistry method of neuron specific enolization enzyme staining. Morphometric analysis for all neuron-specific enolase positive cells was performed by Multifunction Computer Image Analysis System.
RESULTS AND CONCLUSION: After addition of cytarabine at 24 hours of culture, the purity of neurons was more than 90%, well-differentiated cortical neurons accounted for 89.00%, and the area of neuronal body was the largest with the longest synapses. There were more neuron cells with transparent cytoplasm, and large nucleus. The cell body had good refraction and strong stereo sense. The neurons with 3-4 synapses and 3-4 bifurcates formed a good network structure. These results illustrate that although it will be beneficial for the purity of neurons to add cytarabine early in neuron culture process, it will make obvious effect on neuronal differentiation. The highly purified and well-grown cerebral cortical neurons will be obtained after cultured in neurolbasal medium, which cytarabine is added to at 24 hours of culture.

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: Cytarabine, Cerebral Cortex, Neurons, Cells, Cultured, Tissue Engineering

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