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目的:比较不同电转染条件下,真核表达载体转染HL60细胞的效率,筛选得到针对HL60细胞最佳的电转染条件。方法:采用pDsRED-C1真核表达载体,分别在2mm和4mm电转杯中依照不同电转条件对HL60细胞进行转染,根据存活细胞所占比例确定电转参数;在转染48h后,比较不同质粒加入量及二甲基亚砜(dimethylsulfoxide,DMSO)加入电转体系前后的细胞转染阳性率。调整G418筛选浓度,在选定转染条件下进行HL60细胞电转,采用流式细胞技术,细胞化学染色及超微结构观察,分析电转前后HL60细胞的生物学性状。应用相同条件再转染eYFP-C1质粒于HL60细胞,G418筛选后观察荧光表达情况。结果:HL60细胞在2mm和4mm电转杯中的死亡数量随电击强度和脉冲次数的增加而升高,且方形波较回旋波有更强的击穿细胞膜的能力;固定电转参数下,2mm和4mm电转杯中的HL60细胞电转阳性细胞数随加入质粒量的增加呈先升高后下降的趋势,且在相同质粒加入量,2mm电转杯比4mm电转杯有更高的转染效率;在相同电转参数和相同电转杯中,预冷条件下加入DMSO电转时阳性率比不加入DMSO进行电转的阳性率高近13倍;400μg/mlG418是最佳筛选浓度;选定最佳电转条件进行电转,通过筛选,没有发现细胞表面分化抗原CD11b和CD14的表达,细胞形态原始,未见凋亡现象发生。相同条件电转eYFP-C1空载质粒于HL60细胞仍然可以获得很好的转染效果。结论:HL60细胞电转染条件的改良,可以有效提高HL60细胞的电转阳性率,为后续细胞真核表达载体的转染及基因功能研究奠定基础。
OBJECTIVE: To compare the efficiency of eukaryotic expression vector transfected HL60 cells under different electroporation conditions, and to screen the optimal electroporation conditions for HL60 cells. Methods: The pDsRED-C1 eukaryotic expression vector was used to transfect HL60 cells in 2mm and 4mm electric cups respectively according to different electrotransport conditions. The electroporation parameters were determined according to the proportion of surviving cells. After 48h transfection, different plasmids were added And the positive rate of cell transfection before and after adding dimethylsulfoxide (DMSO) to the electrotransposition system. After adjusting the concentration of G418, HL60 cells were electroporated under selected transfection conditions, and the biological characteristics of HL60 cells before and after electroporation were analyzed by flow cytometry, cytochemical staining and ultrastructure observation. The same conditions were transfected with eYFP-C1 plasmid in HL60 cells, G418 screening fluorescence expression was observed. Results: The number of death of HL60 cells in 2mm and 4mm electric cups increased with the increase of the electric shock intensity and the number of pulses, and the square wave had more ability of penetrating the cell membrane than the gyroc wave; The number of electropositive cells of HL60 cells in the rotor increased first and then decreased with the increase of the amount of plasmids, and the transfection efficiency of the 2mm rotor was higher than that of the 4mm rotor in the same amount of plasmid. In the parameters and the same cup, the positive rate of DMSO electroporation was nearly 13 times higher than that of DMSO without pretreatment under the condition of precooling; the optimal screening concentration was 400μg / ml G418; Screening, did not find the cell surface differentiation antigen CD11b and CD14 expression, the original cell morphology, no apoptosis occurred. Under the same conditions electroporation eYFP-C1 empty plasmid in HL60 cells can still get a good transfection effect. CONCLUSION: The improvement of electroporation condition of HL60 cells can effectively increase the positive rate of electroporation in HL60 cells, which lays the foundation for the transfection and gene function study of eukaryotic expression vectors in HL60 cells.