新生大鼠培养海马神经元的双重转染和活细胞成像

Double transfection and live cell imaging of cultured hippocampal neurons in neonatal rat

目的建立新生大鼠海马神经元的双重转染和活细胞成像方法。方法出生24h内的SD大鼠海马神经元原代培养,接种后第5天双重转染表达绿色荧光蛋白的GFP-GluR1和红色荧光的DsRed,活细胞成像观察转染后细胞情况。结果转染48h后神经元基本稳定,发绿色荧光,胞体大呈多边,立体感强,突起长且连接密集;可以在不同时间点定位观察同一部位;双重转染GluR1-GFP和DsRed质粒48h后,可在倒置显微镜下观察到荧光,GluR1-GFP表达于树突,DsRed表达于树突棘。结论获得了稳定可行的神经元双重转染技术和长时程在不同时间点定位追踪观察培养神经元的成像方法。

Objective To establish hippocampal neurons double transfection and live cell imaging methods. Methods The hippocampal neurons of SD rat birth within 24 h were cultured,then doubly transfected green fluorescent expressing GFP-GluR1 and red fluorescent expressing DsRed plasmids the fifth day after planting. Live cell imaging was applied to observe transfected neurons. Results Neurons were basically stable, expressing green fluorescence after 48 h transfection. Cell body was polygon with strong three-dimensional sense. Protrusions were long and con-nected densely. This method enable us to observe the same site of neurons at a different time point;doubly transfected with GluR1 -GFP and DsRed plasmids,fluorescence can be detected under an inverted microscope after 48 h. GluR1-GFP was expressed in dendritic and DsRed was expressed in dendritic spines. Conclusion We successfully acquired a stable viable doubly transfection technology and a live cell imaging method to observe cultured neurons at different time points for a long-term.