文章摘要
鳜脑神经元的原代培养与鉴定
Primary Cell Culture and Identification of brain neurons in Chinese perch (Siniperca chuatsi)
投稿时间:2019-01-15  修订日期:2019-05-06
DOI:
中文关键词:     神经元细胞  原代培养
英文关键词: Chinese perch  Brain  Neurons cell  Primitive culture
基金项目:国家自然科学基金面上项目(31772822);国家自然科学青年基金(31602131),
作者单位E-mail
石林杰 1.华中农业大学水产学院华中农业大学鳜鱼研究中心武汉4300702. 农业部鳜鱼育种创新基地农业部淡水生物繁育重点实验室武汉430070 1451644883@qq.com 
梁旭方 1.华中农业大学水产学院华中农业大学鳜鱼研究中心武汉4300702. 农业部鳜鱼育种创新基地农业部淡水生物繁育重点实验室武汉430070 xfliang@mail.hzau.edu.cn 
何珊 1.华中农业大学水产学院华中农业大学鳜鱼研究中心武汉4300702. 农业部鳜鱼育种创新基地农业部淡水生物繁育重点实验室武汉430070  
彭建 1.华中农业大学水产学院华中农业大学鳜鱼研究中心武汉4300702. 农业部鳜鱼育种创新基地农业部淡水生物繁育重点实验室武汉430070  
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中文摘要:
      目前鱼类神经细胞模型极少,物种间的差异性使鳜在细胞水平上的研究具有极大的局限性,为建立一种简单易行的鳜脑神经元细胞模型进一步深入研究鳜神经系统。本研究取3月龄鳜,联合胶原酶消化法和机械吹打法分离出鳜脑细胞,用含20% FBS的L15培养液制成细胞悬液接种在细胞培养瓶内,于28℃无CO2培养箱中培养,3天后更换培养基,待细胞贴壁长满瓶底后进行传代培养;采用NeuN和b-tublin免疫荧光细胞化学技术鉴定鳜脑细胞神经元纯度。结果显示:鳜脑细胞分离培养2天后贴壁状态较好,随着时间延长,贴壁细胞增多,细胞突起相互连接,培养5天后神经元胞体丰满,细胞数量明显增多,突起相互间形成了明显的神经网络。经NeuN和b-tublin免疫荧光细胞化学技术鉴定鳜脑神经元细胞纯度可达95%以上。研究表明该鳜脑细胞的分离培养方法简单易行,可获得高纯度的鳜脑神经元细胞。
英文摘要:
      The diversity of species makes the research on cell level of Chinese perch extremely limited. Establishing a simple and feasible method for primary culture of brain neurons from Chinese perch (Siniperca chuatsi) in vitro is beneficial to further study on fish nervous system. Our laboratory combined with the usual method of cell culture, Chinese perch brain neruons (born 3 months) were isolated by collagenase digestion and mechanical blow. L15+20%FBS suspended brain neruons were inoculated on the cell culture vessels, in 28℃ without CO2 incubator. The medium was replaced after 3 days. Subculture was carried out after the cells covered with the bottom of the cell culture vessels. The morphological changes of the neurons were observed under inverted phase-contrast microscope; Immunofluorescence staining for NeuN or β-tublin was performed to identify the purity of neurons. The results showed that the cells began to adhere to the culture bottle and develop small neurites and form network gradually after Primary culture for 2 days. Up to the 5th day, many neurites extended to form dense network and Soma of neurons became well. Fluorescence staining with NeuN or b-tublin showed that the purity of neurons can reach above 95%. The present protocol is a simple and efficient method for culturing brain neurons of chinese perch with high purity, Which is of great significance for the fine study of fish growth and development, expression regulation of various receptors and proteins, cell apoptosis and cell signal transduction.
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