急性早幼粒白血病HL60细胞电转染条件的优化

A Suitable Method for Electroporation of Human Acute-premyelocyte HL60 Cell Line

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。

Objectives：To compare the efficiency of HL60 cellular transfection by eukaryotic expressing vector using different methods,and then the most suitable way for transfection could be screened out.Methods：Transfected the empty eukaryotic expressing vector into HL60 cells using 2mm and 4mm electroporating cups respectively using different instrumental parameter,following which the parameter could be ascertained based on the survival rate of HL60 cells.The comparison of transfection efficiency after 48h cellular culture was performed between groups with different plasmid addition and DMSO（dimethyl sulfoxide） supplement.The G418 was employed to screen out positive clones,and its concentration was chosen based on positive rate analysis.At last,pDsRED-C1 empty expressing vector was transfected into HL60 cells following the methods above,and cellular biocharacters could be observed using flow cytometry（FCM）,cellular chemical staining and transmission electron microscope.What’s more,the same transfecting procedure was used to transfected another empty eukaryotic expressing vector eYFP into HL60 cells.Results：The death rate of HL60 cells was increased in line with the elevation of intensity of electric shock and frequency of pulse.Square wave was stronger in electroporation than convolution wave.Along with the addition of plasmid,the number of positive transfected HL60 cells was up-regulated at first,and down-regulated subsequently with the constant tansfected parameter.Moreover,the positive transfected HL60 cells was much more in 2mm electroporated cup than 4mm electroporated cup under the same plasmid addition.The supplyment of DMSO in refrigerated transfection system could significantly improve the positive rate of transfection about nearly 13 times.400μg/ml G418 was choosen as the best screening drug concentration.After cellular electroporation,the cellular surface antigen CD11b and CD14 could not be detected,and that,the apoptosis could not be found by observation of cellular morphology and ultramicrostructure.Same procedure was successfully used in the transfection of eYFP-C1 into HL60 cells.Conclusion：The modified electroporating procedure could significantly promote the positive rate of cellular transfection,which would lay the foundation of researchers’ following experiments.