脂质体介导pAcGFP-bFADD重组质粒转染Hela细胞条件的优化  

Optimization of Transfection Conditions for Tranfecting pAcGFP-bFADD Recombinant Plasmid to Hela Cell Mediated by Lipofectamine 2000

作　　者：杨润军[1,2] 李武峰[3] 张路培[1] 许尚忠[1,2] 李俊雅[1] 高雪[1] 陈金宝[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]西北农林科技大学动物科技学院,杨凌712100 [3]山西农业大学生命科学院,太谷030801

出　　处：《农业生物技术学报》2009年第5期786-791,共6页Journal of Agricultural Biotechnology

基　　金："十一五"国家高技术研究发展计划(863)(No.2006AA107Z197);"十一五"国家科技支撑计划重大项目(No.2006BAD01A10)资助

摘　　要：用脂质体lipofectamine 2000介导携带有AcGFP报告基因的pAcGFP-bFADD质粒转染Hela细胞,并通过调整质粒DNA、脂质体的浓度及转染时间来优化转染条件,转染后12h在荧光显微镜下计数阳性细胞,MTT法检测各转染条件下Hela细胞的活性。结果表明,将2.4μg的pAcGFP-bFADD质粒与3.0μL lipofectamine 2000制备转染复合物与Hela细胞作用6h可以获得62.3%的转染效率,并且保持较高的细胞存活率(83%)。当质粒DNA与脂质体的量增加至3.2μg和6.0μL时,转染复合物对细胞的毒性作用明显增强,对细胞的抑制率可达42.98%,与对照组相比,差异极显著(P<0.01);条件优化后AcGFP-bFADD融合基因于转染后72h达到表达高峰,绿色荧光阳性细胞所占比率达到64%。pAcGFP-bFADD plasmid with AcGFP report gene was transfected to Hela cell mediated by lipofectamine 2000, and the transfection condition was optimized by adjusting transfection time and concentrations of plasmid DNA and liposome.Twelve hours after transfected, positive cells count was made under fluorescence microscope, and the cell viability was detected by MTT.Results indicated that perfect cell transfection efficiency（62.3% ） was obtained on the condition of the complex of 2.4 μg pAcGFP-bFADD and 3.0 μL lipofectamine 2000 transfecting Hela cell after 6 h, meanwhile, its cell viability could reach 83%.When the concentration of plasmid DNA and liposome increased to 3.2 μg and 6.0 μL respectively, the complex would bring higher toxicity, the inhibition rate reached 42.98%, the difference was extremely significant（P 〈0.01） compared with control group.After condition optimization, AcGFP-bFADD fusion gene got the expression peak 72 h after transfection.The green fluorescent positive cells ratio could reach 64%.

关 键 词：转染 lipofectamine2000 重组质粒 HELA细胞 

分 类 号：S188[农业科学—农业基础科学]