脂质体介导法转染mIMCD-3细胞的可行性及最佳转染条件

Optimization of transfection efficiency of mIMCD-3 cells mediated by liposome

背景:近年来阳离子脂质体成为基因转移较为常用的载体,但应用脂质体对mIMCD-3细胞株进行转染的相关报道较少。目的:探讨脂质体法转染mIMCD-3细胞株的可行性,并通过优化脂质体转染效率的影响因素,获得mIMCD-3的最佳转染条件。方法:以绿色荧光蛋白作为报告基因,采用脂质体LipofectamineTM2000包裹pIRES2-EGFP质粒转染mIMCD-3细胞,分别按照细胞接种密度、DNA用量、DNA与脂质体的比例、不同的质粒抽提方法、血清有无设定分组,观察以上因素对mIMCD-3细胞转染效率的影响。结果与结论:采用脂质体LipofectamineTM 2000转染mIMCD-3细胞,在2×108L-1细胞接种浓度、1μgDNA用量、1:3的DNA与脂质体比例时,转染效率最高。采用美国OMEGA公司生产的E.Z.N.A. Endo-Free Plasmid Mini Kit抽提的质粒能获得更高的转染效率。转染前漂洗细胞后换入无血清培养基比有血清组转染效率高(P<0.01),而转染后6h加入血清不影响转染效率。结果显示,脂质体法转染mIMCD-3细胞株是可行的,通过优化脂质体转染效率的影响因素,获得了mIMCD-3的最佳转染条件,可作为有关研究或应用的参考。

BACKGROUND：Recently,cationic liposome becomes the commonly used vehicles for gene transfer.However,few study reports transfection of mIMCD-3 cell lines mediated by liposome.OBJECTIVE：To approach the feasibility of transfection of mIMCD-3 mediated by liposome and to obtain the optimized transfection condition for mIMCD-3.METHODS：Using green fluorescent protein as reporter gene,the plasmid pIRES2-EGFP was transfected into mIMCD-3 cells with Lipofectamine^TM 2000.To set different groups according to cell density,DNA amount,ratio of DNA and liposome,plasmid extraction methods and serum,and to observe the influences on transfection efficiency by these different factors.RESULTS AND CONCLUSION：The highest transfection efficiency was achieved with the following optimized conditions：2×10^5/L cells,1 g DNA,1：3 ratio of DNA and liposome.Extracting plasmid by E.Z.N.A.Endo-Free Plasmid Mini Kit produced by American OMEGA company obtained higher transfection efficiency.Compared with serum medium,cells culutred with serum-free medium obtained higher transfection efficiency before transfection（P〈0.01）,but it had no effect at 6 hours after transfection.Transfection of mIMCD-3 mediated by liposome is feasible,and the optimized transfection condition for mIMCD-3 is obtained.These results may be used as the reference for other studies.