一种低密度及高纯度胎鼠原代海马神经元培养方法的优化及鉴定  

Optimization and identification of a low density and high purity method for primary hippocampal neuron culture from fetal rats

作　　者：苏芃 王兴仪 梁景岩 熊天庆 SU Peng;WANG Xing-yi;LIANG Jing-yan;XIONG Tian-qing(Institute of Translational Medicine,Medical College,Yangzhou University,Jiangsu Yangzhou 225001,China;Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases,Yangzhou University,Jiangsu Yangzhou 225001,China)

机构地区：[1]扬州大学医学院转化医学研究院,江苏扬州225001 [2]江苏省中西医结合老年病防治重点实验室,江苏扬州225001

出　　处：《解剖学报》2024年第1期113-119,共7页Acta Anatomica Sinica

基　　金：江苏省研究生科研与实践创新计划项目(KYCX22_3562);中国博士后科学基金(2022M712689)。

摘　　要：目的通过将海马、皮层细胞共培养的方式建立一种低密度、高纯度、高稳定性的大鼠胎鼠原代海马神经元体外培养方法,以获取纯度更高、活力更好的原代海马神经元。方法分离孕16~18 d SD大鼠胎鼠的海马和皮层并分别剪碎,经0.125%胰蛋白酶消化后用200目细胞筛过滤,将获得的细胞悬液以包围的形式分别接种于培养板的内层和外环上,于含10%马血清的DMEM/F12培养基中进行共同培养,4~6 h细胞贴壁后更换培养基为维持培养基培养(Neurobasal培养基+2%B27+0.5 mmol/L谷氨酰胺)。通过CCK-8试剂盒检测细胞活力,利用免疫荧光染色检测海马神经元纯度。结果海马神经元在5 d后形成纵横交错的神经网络,生长状态较好,经鉴定海马神经元的纯度高达98%,且能够稳定存活3周。结论利用海马、皮层细胞共培养的方法可以获取高纯度、高活性、高存活率、高稳定性的胎鼠原代海马神经元,可为神经系统中海马神经元相关疾病研究提供一定的实验条件。Objective To establish a low density,high purity and high stability in vitro culture method of primary hippocampal neurons of fetal rats by co-culturing hippocampal and cortical cells,so as to obtain higher purity and better vitality of primary hippocampal neurons disease.Methods The fetal rat hippocampal tissue was isolated from 16-18 days pregnant SD rats,then cut and digested by 0.125%trypsin.The obtained cell suspension was filtered by 200 mesh cell sieve,and then the obtained cell suspensions were then inoculated into the inner layer and outer ring of the culture plate in a surrounding form.They were co-cultured in DMEM/F12 medium containing 10%horse serum.After 4-6 hours of cell adhesion,the culture medium was changed to maintenance medium(Neurobasal+2% B27+0.5 mmol/L glutamine).Then the cell viability was detected with CCK-8 kit and the purity of hippocampal neurons was detected by immunofluorescent staining.Results Hippocampal neurons grew well and formed crisscross neural networks after 5 days.And it could survive for 3 weeks.The purity of hippocampal neurons was up to 98%.Conclusion The method of co-culturing hippocampal and cortical cells can obtain high-purity,high activity,high survival rate,and high stability primary hippocampal neurons from fetal rats,which can provide certain experimental conditions for the study of hippocampal neuron related diseases in the nervous system and is worthy of promotion and application.

关 键 词：海马神经元 原代培养 低密度 免疫荧光 大鼠 

分 类 号：R332[医药卫生—人体生理学]