贵州医科大学学报

2020, v.45;No.239(08) 894-898

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海马神经元原代细胞培养方法的改良
Improvement of Primary Cell Culture Method for Hippocampal Neurons

高超;杜贵琴;许永劼;朱金凤;潘卫;李兴;
GAO Chao;DU Guiqin;XU Yongjie;ZHU Jinfeng;PAN Wei;LI Xing;Department of Medical Laboratory,Guizhou Medical University;Guizhou Prenatal Diagnosis Center,the Affiliated Hospital of Guizhou Medical University;Key Laboratory of Environmental Pollution Monitoring and Disease Control of Ministry of Education,Guizhou Medical University;School of Basic Medical Sciences,Guizhou University of Traditional Chinese Medicine;

摘要(Abstract):

目的:旨在建立一种简单、稳定的SD乳鼠海马神经元的分离及原代培养方法。方法:选用出生24 h内的SD乳鼠,在数码显微镜下分离双侧海马体,剥离去除海马体上的包膜血管,剪碎吹打海马组织、胰酶消化后,获得细胞悬液,用含胎牛血清的DMEM高糖型种植培养基培养8 h后,再用添加B27的Neuralbasal维持型培养基行原代培养,第7天采用神经元特异性烯醇化酶(NSE)免疫组织化学染色鉴定神经元纯度,CCK-8法检测海马神经元细胞活力。结果:提取的海马神经元细胞形态完整,胶质细胞少,基本无杂质,生长状态良好,胞体丰满,培养的海马神经元之间能形成密集的网络结构; NSE免疫组化鉴定培养的海马神经元纯度达90%,CCK8显示培养第1~6天培养的海马神经元活性逐渐增强,第7天达峰值。结论:本方法能够提高海马神经元细胞纯度以及细胞活性,是一种稳定高效的海马神经元原代培养方法。
Objective: To establish a simple and stable method for isolating and culturing primary hippocampal neurons from SD suckling rats within 24 hours after birth. Methods: The bilateral hippocampus was taken from the SD suckling rats within 24 hours after birth under a digital microscope. The capsular vessels on the hippocampus were peeled off,the hippocampal tissue was minced,digested with trypsin and centrifuged to obtain the cell suspension. After the resultant cells were cultured with high-glucose DMEM supplemented with fetal bovine serum for 8 hrs,these cells were cultured with Neuralbasal maintenance medium supplemented with B27. Immunohistochemical( IHC) staining of neuronal specific enolase( NSE) was used to identify neuron purity on day 7. CCK-8 assay was used to measure the viability of primary hippocampal neurons. Results: The isolated hippocampal neurons exhibited complete cell morphology and full cell bodies and good growth status.They were highly pure with few glial cells. A dense network structure was constructed between the cultured hippocampal neurons. IHC staining of NSE showed that the purity of isolated primary neurons reached 90%. CCK8 assay showed that the viability of these neurons gradually increased from days 1 to 6 and reached a peak on day 7. Conclusion: This method can improve the purity and viability of isolated hippocampal neurons,and is a stable and efficient method to culture primary hippocampal neurons.

关键词(KeyWords): 海马;神经元;细胞,培养的;糖尿病;免疫组织化学;原代细胞
hippocampus;neurons;cells,cultured;diabetes mellitus;immunohistochemistry;primary cells

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81560138,81560720);; 贵州省科技计划项目[黔科合支撑(2019)2802号];; 贵州省教育厅创新群体重大研究项目[黔科合KY字(2018)021]

作者(Author): 高超;杜贵琴;许永劼;朱金凤;潘卫;李兴;
GAO Chao;DU Guiqin;XU Yongjie;ZHU Jinfeng;PAN Wei;LI Xing;Department of Medical Laboratory,Guizhou Medical University;Guizhou Prenatal Diagnosis Center,the Affiliated Hospital of Guizhou Medical University;Key Laboratory of Environmental Pollution Monitoring and Disease Control of Ministry of Education,Guizhou Medical University;School of Basic Medical Sciences,Guizhou University of Traditional Chinese Medicine;

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DOI: 10.19367/j.cnki.2096-8388.2020.08.005

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