摘要
目的优化HepG2细胞电转染条件,进一步提高HepG2细胞的转染效率。方法采用电穿孔方法将pcDNA3.1-EGFP导入HepG2细胞中,在500μL电转染体系中,在不同电场强度、细胞数目、脉冲频率、脉冲时间、电转质粒数目、电转缓冲液、培养基血清浓度条件下,分别将pcDNA3.1-EGFP质粒电转染HepG2细胞,检测不同条件下细胞存活率和转染率。结果电转前4℃孵育电转体系混合液10 min,方波电转条件在1个电脉冲、电压270V、细胞数为2×106个、质粒20μg、脉冲时间20 ms、电转缓冲液为优化缓冲液、电转后置于37℃、含15%FBS的DMEM高糖培养基中培养48 h,可获得高转染率(60.68±1.87)%。结论优化电穿孔法的电转染条件能够有效提高HepG2细胞的电转染效率。本研究为外源基因电转染HepG2细胞提供了可靠的试验参数。
Objective To optimize the electransfection parameters of HepG2 ceils and further to improve the transfec- tion efficiency of HepG2 ceils. Methods We electransfected pcDNA3.1-enhanced green fluorescent protein （pcDNA3.1- EGFP） into HepG2 cells by electroporation apparatus, and HepG2 cells were transfected in 500 trL transfection volume un- der different electric field intensity, cell number, pulse frequency, impulse time, plasmid concentration, electroporation medium and outgrowth medium. Then, the survival rates and transfection rates were calculated. Results 500μL mix- tures were incubated at 4 ℃, the square-wave electransfection was optimized with the best transfection efficiency of （60.68 ± 1.87） % under the following conditions ： the electric voltage 270 V, one square-wave, 20 ms time, 1 Hz frequency, the cell number 2×10^6/mL, the EGFP plasmid concentration 20μg, electroporation buffers as the optimum culture medium, and mixtures were incubated in 15% FBS outgrowth medium for 48 h after the pulse. Conclusions By optimizing the electransfection conditions, the electransfection rate of HepG2 cells is efficiently improved. This study provides the reliable parameter basis for exogenous gene transfection of HepG2 cells.
出处
《山东医药》
CAS
2013年第42期1-4,8,共5页
Shandong Medical Journal
基金
国家"十二五"科技支撑计划项目(2013BAI05B03)
