利于树突棘形态学观察的原代海马神经元转染方法的优化  

Optimization of transfection method for primary hippocampal neurons benefiting for morphological observation of dendritic spines

作　　者：李莎[1,2] 司尧 冯宝峰 张沂洲[1,2] 崔慧先 Li Sha;Si Yao;Feng Baofeng;Zhang Yizhou;Cui Huixian(Department of Anatomy,Hebei Medical University,Shijiazhuang 050017,China;Neuroscience Research Center,Hebei Medical University,Shijiazhuang 050017,China)

机构地区：[1]河北医科大学人体解剖学教研室,石家庄0050017 [2]河北医科大学神经科学研究中心,石家庄0050017

出　　处：《解剖学杂志》2018年第5期532-534,557,共4页Chinese Journal of Anatomy

基　　金：国家自然科学基金(31471145;31701047);河北省高等学校科学技术研究重点项目(ZD2014047);河北省教育厅青年拔尖人才项目(BJ2017007)

摘　　要：目的:为有助于观察树突棘的变化,探索出较为理想的原代大鼠海马神经元转染方法。方法:应用Lipofectamine2000和慢病毒2种转染方法对体外培养8d的SD胎鼠海马神经元进行绿色荧光蛋白(GFP)质粒转染,观察海马神经元转染效率和荧光表达情况,并用台盼蓝染色检测神经元的存活率;采用优选出的转染方法对体外培养8d的海马神经元进行转染,观察转染后培养至10、15、20d和25d不同时间海马神经元树突棘的生长发育情况;观察8、12d和16d不同时间转染对海马神经元树突棘形态学观察的影响。结果:对于树突棘形态学观察而言,Lipofectamine 2000转染效果显得更为优越;海马神经元培养至20d时,树突棘发育较成熟;12d时转染是进行树突棘形态学观察转染的最佳时间。结论:用Lipofectamine 2000转染GFP方法对培养12d的神经元进行转染,培养至20d时树突棘形态学观察效果良好。Objective:To explore an ideal method for primary transfection of rat hippocampal neurons so as to observe the changes of dendritic spines.Methods:The green fluorescent protein(GFP)plasmid was transfected into hippocampal neurons of SD fetal rats by Lipofectamine 2000 and lentivirus transfection for 8 days in vitro.The transfection efficiency and fluorescence expression of hippocampal neurons were observed,and the survival rate of neurons was detected by trypan blue staining.Then,the cultured hippocampal neurons were transfected with the optimized transfection method for 8 days in vitro.The growth and development of the dendritic spines of the cultured hippocampal neurons were observed at 10,15,20 days and 25 days after transfection.Finally,the effects of 8,12 days and 16 days of transfection on the morphology of dendritic spines of hippocampal neurons were observed.Results:For morphological observation of dendritic spines,the transfection efficiency of Lipofectamine 2000 was superior.When the hippocampal neurons were cultured to 20 days,the development of dendritic spines was more mature.Transfection at 12 days was the best time to observe the morphology of dendritic spines.Conclusion:The primary hippocampal neurons transfected with Lipofectamine 2000 at 12 days and observed at 20 days have good morphology of dendritic spines.

关 键 词：海马神经元 转染 LIPOFECTAMINE 2000 慢病毒 树突棘 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]