海马神经元原代培养方法

Primary culture method of hippocampal neurons

目的：掌握海马神经元的有效培养方法和细胞保存培养方法,利于细胞模型的建立。方法：取出出生1 d的SD大鼠海马,联合机械吹打法和胰酶消化法分离海马细胞,加入DMEM/F12＋10%FBS制备细胞悬液,接种在玻璃培养瓶内1～2 d,收集上清细胞液、离心、重悬细胞;再接种在培养板上,培养1 d后,全液更换为Neurobasal＋2%B27,以后每3 d半量换液。利用倒置相差显微镜观察细胞形态变化;利用NF-200、Neu-N抗体免疫荧光、免疫组化技术鉴定海马神经元;利用96孔板培养细胞,计数每个孔海马细胞比例;利用MTT法测定细胞活性。结果：接种在培养板上第1 d细胞贴壁,第3～4 d细胞长出突触,第7～8d细胞突触生长迅速,第11～13 d细胞交织成网状,第15～20 d细胞生长旺盛,20 d后细胞形态开始改变。免疫荧光技术鉴定细胞突触呈红色,细胞核呈绿色;免疫组化技术鉴定突触和胞核都呈棕黄色。细胞纯度95～97.5%,细胞活性以15～17 d最高（93.94～95.13%）,与其他时间点比较,P〈0.01。结论：该方法简便可行,节约取材及时间,可获得纯度高、活性好的海马细胞,为后续研究提供实验基础。

Objective： To establish an effective culture method of hippocampal neurons and preservable cells for the establishiment of cell models. Methods： SD rat hippocampal neruons（born 1 day） were isolated by mechanical blow and pancreatic enzyme digestion. DMEM/F12＋10%FBS suspended hippocampal neurons were inoculated on the glass culture vessels（1～ 2 days）. The upper cell suspension was collected,centrifugated, resuspended and inoculated on orifice plates for one day. The medium was replaced by Neurobasal ＋ 2%B27 every 3 days. Inverted phase contrast microscope was used to observe cell morphological changse. Hippocampus neuronal identification and cell activity were detected with immunocytochemistry and MTT. Results： Hippocampal neurons were adherent to orifice plates on the first day. The cell synapses grew on the 3th ～ 4th day. The cell synapses reticulate on the 11th ～ 13th day. The cells grew especially strong on the 15th ～ 20th day and after the 20th day there was cell apoptosis. The cell synapse was red and nucleus was green that was identified with immunofluorescent staining. The cell synapse and nucleus were brownish yellow with immunohistochemistry. The neruon purity was 95% ～ 97.5%.The cell activity was the highest in the 15th ～17th day（93.94% ～ 95.13%） compared with other time growps（P 0.01）. Conclusion： Our culture method is economical and can save a lot of time with simplicility and feasibity. The high purity and activity of hippocampal neurons provide the experimental basis for further study.