流式细胞术优化MDCK细胞基因转染效率的研究  

Optimization and Researches of Gene Transfection Efficiency of MDCK Cells by Flow Cytometry

作　　者：王家敏[1,2] 王美皓 马玉梅 靳冬武 赵彩红 李自良[1,2] 乔自林 马忠仁[1] WANG Jia-min;WANG Mei-hao;MA Yu-mei;JIN Dong-wu;ZHAO Cai-hong;LI Zi-liang;QIAO Zi-lin;MA Zhong-ren(Gansu Tech Innovation Center of Animal Cell,Biomedical Research Center,Northwest Minzu University,Lanzhou,730030,China;Life Science and Engineering College,Northwest Minzu University,Lanzhou,730030,China;Lanzhou Lark Biotechnology co.LTD,Lanzhou,730010,China)

机构地区：[1]西北民族大学生物医学研究中心甘肃省动物细胞技术创新中心,甘肃兰州730030 [2]西北民族大学生命科学与工程学院,甘肃兰州730030 [3]兰州百灵生物技术有限公司,甘肃兰州730010

出　　处：《西北民族大学学报（自然科学版）》2020年第1期16-20,共5页Journal of Northwest Minzu University(Natural Science)

基　　金：国家科技重大专项重大新药创制项目(2015ZX09102016);西北民族大学动物细胞培养与代谢工程创新团队(31920190004);甘肃省重点研发计划(18YF1FA121).

摘　　要：为探究脂质体介导的MDCK细胞基因转染效率,并获得最佳的优化方案,本研究选取不同配比的质粒和脂质体浓度,将p-EGFP-C1质粒转入MDCK细胞.转染后36 h,利用台盼蓝染色法检测细胞活率,流式细胞术检测细胞中增强型绿色荧光蛋白(EGFP)的表达情况和转染效率,获得优化的转染条件.结果发现,转染后MDCK细胞,实验组细胞活力与阴性对照组差异不显著(P>0.05).当质粒(μg)∶脂质体(μL)浓度配比为0.5∶1.0、0.5∶1.25、0.5∶1.5时,转染率达到了较高水平,分别为(23.4±3.4)%、(24.5±4.5)%、(25.2±3.7)%,从经济因素考虑,脂质体介导的MDCK细胞基因转染质粒(μg)与脂质体(μL)的最佳配比浓度为0.5∶1.0.这为MDCK细胞用于基因转染提供了一定的理论基础.In order to explore the efficiency of liposome-mediated MDCK cell gene transfection and obtain a better optimization scheme.In this study,plasmid and liposome of different proportions were selected to transfer p-EGFP-C1 plasmid into MDCK cells.The expression and transfection efficiency of enhanced green fluorescent protein(EGFP)was detected by flow cytometry at 36h after transfection.The cell viability was determined by trypan blue staining to obtain the optimal transfection conditions.It was found that after transfection,the cell activity of MDCK cells in the experimental group was not significantly different from that of the negative control group.Transfection rate analysis showed that when the concentration of the plasmid(μg)and liposome(μg)was 0.5∶1.0,0.5∶1.25 and 0.5∶1.5.The transfection rate reached a very high level about 23.4±3.4%,24.5±4.5%and 25.2±3.7%,respectively.Considering economic factor,the optimal concentration of plasmid and liposome in MDCK cell gene transfection was 0.5∶1.0.This provides a theoretical basis for the use of MDCK cells in gene transfection.

关 键 词：MDCK细胞 基因转染 脂质体 流式细胞术 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]