小鼠背根神经节取材与电穿孔转染的条件优化  

Optimization of Conditions for Transfection of Mouse Dorsal Root Ganglia and Electroporation

作　　者：韦善文 马艳霞 WEI Shan-wen;MA Yan-xia(Institute of Orthopedics,School of Medicine,Soochow University,Suzhou,Jiangsu,215007,China)

机构地区：[1]苏州大学医学部骨科研究所,江苏苏州215007

出　　处：《中国血液流变学杂志》2018年第4期377-379,F0002,共4页Chinese Journal of Hemorheology

摘　　要：目的以小鼠背根神经节(dorsal root ganglion, DRG )细胞为工具,绿色荧光蛋白为报告基因来优化电穿孔转染条件。方法分别用传统从背部取材DRG的方法和改良的从腹侧取材DRG的方法进行DRG取材,比较两种方法取材相同数量DRG所用时间的差异,进行统计学分析。以及通过控制不同的电压条件,将表达绿色荧光蛋白的质粒电穿孔导入小鼠DRG细胞中,3d后观察比较神经元的转染效率,荧光显微镜观察统计确定最佳的电转染效率。结果通常传统方法取出6个形态完整的L3-L5节段的DRG (共6个/只小鼠),平均用时2 min/DRG,而掌握改良的方法后,取得6个形态完整的L3.L5节段的DRG,平均用时1.3 min/DRGo与传统方法相比差异具有统计学意义(P<0.05).110V电压,10 ms脉冲时间的转染条件下,小鼠DRG神经元可以达到20%的转染效率。结论经过改良后的小鼠DRG取材方法明显改善了所取DRG的完整性以及加快了取材DRG的速度,提高了DRG神经元培养的成活率,为后续实验提供了高质量的材料。而电穿孔条件的优化提高了小鼠背根神经节神经元的电转染效率。Objective To optimize the electroporation transfection conditions by using mouse dorsal root ganglion (DRG) cells as a tool and green fluorescent protein as a reporter gene. Methods The DRG method and the modified DRG method were used to obtain DRG. The differences in time between the two methods were compared for statistical analysis. And by controlling different voltage conditions, the plasmid expressing green fluorescent protein was introduced into the DRG sensory neurons of mice, and the transfection efficiency of the neurons was observed after 3 days. The best electrotransfection efficiency was determined by fluorescence microscopy. Results 6 morphologically intact L3-L5 segments of DRG (6 DRG/mouse) were taken by conventional methods, and the average time was 2 min/DRG. After mastering the improved method, 6 intact L3- L5 samples were obtained. The segmental DRG averaged 1.3 min/DRG. Compared with the traditional method, the difference is statistically significant (Pc 0.05). Under the transfection conditions of 110 V voltage and 10 ms pulse time, the mouse DRG neurons can achieve 20% transfection efficiency. Conclusion The improved DRG method has improved the integrity of DRG and accelerated the speed of DRG, and improved the survival rate of DRG neurons. High quality materials were provided for subsequent experiments. The optimization of electroporation conditions improved the electrical transfection efficiency of mouse DRG neurons.

关 键 词：DRG取材 电穿孔 基因转染 

分 类 号：R725.6[医药卫生—儿科]