FuGENE 6 Transfection Reagent及pEGFP-N1-HSF1转染JEG-3细胞条件的优化  

Optimization of Condition for Transfection of JEG-3 Cells with FuGENE~? 6 Transfection Reagent

作　　者：杨友辉 刘香香 吴琼[1] 薛维娜[1] 刘亭[1] 陈一飞 

机构地区：[1]贵州医科大学,贵州省药物制剂重点实验室,贵阳550004 [2]上海药品审评核查中心,上海201203 [3]上海交通大学医学院附属国际和平妇幼保健院,上海200030

出　　处：《贵州医科大学学报》2017年第12期1384-1388,共5页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81401216,81760699);贵州省科学基金[黔科合J字(2014)2018号];贵阳市科技计划(联合基金)项目[(20161001)03];贵州省科学技术厅人才团队项目[黔科合平台人才(2016)5613\5677];贵州省教育厅项目[黔教合协同创新字(2013)04]

摘　　要：目的:优化FuGENE~?6 Transfection Reagent(FuGENE)转染质粒pEGFP-N1-HSF1到人绒毛膜癌细胞JEG-3的条件。方法:利用DNA延滞实验考察pEGFP-N1-HSF1质粒与FuGENE试剂的结合力,用不同剂量的FuGENE试剂和质粒转染JEG-3细胞,通过倒置荧光显微镜和血球计数法计算其转染效率,筛选出最佳的转染条件并进行转染,通过qRT-PCR检测HSF1 mRNA的水平。结果:DNA延滞实验显示FuGENE和pEGFP-N1-HSF1质粒具有较高的结合力,当质粒量一定时,随着FuGENE量的增加,其转染效率先增加后降低;当FuGENE和质粒比例为2∶1(μL∶μg)时,转染效率最高,达(39.05±1.19)%;当固定转染比例时,随着转染剂量的增加,其转染效率先增加后降低,当转染剂量为250μL时,转染效率最高,达(42.98±2.56)%;qRT-PCR结果显示,转染组HSF1 mRNA的水平较对照组有显著上调。结论:在转染比例为2∶1,转染剂量为250μL时,对JEG-3细胞有最佳的转染效率。Objective： To optimize the transfections for pEGFP-N1-HSF1 into JEG-3 cells mediated by FuGENE~？6 Transfection Reagent（ FuGENE）. DNA arrearage assay was firstly applied to determine the capability of FuGENE with pEGFP-N1-HSF1. JEG-3 cells were used as the resaearch objects and transfection experiments were carried out at different concentrations of FuGENE reagent and plasmid. The transfection efficiency was calculated by the inverted fluorescence microscope and blood cells count method,and the optimum transfection conditions were screened. The cells were transfected under the optimized condition,and the mRNA levels of HSF1 in the cells were identified by qRT-PCR.Results： DNA delay experiments showed that FuGENE and pEGFP-N1-HSF1 plasmid were strongly bonded. When the amount of plasmid was determined,the transfection efficiency increased first and then decreased with the increase of FuGENE concentration. When the ratio of FuGENE and plasmid was 2∶ 1（ 4 μL∶ 2 μg）,the highest transfection efficiency was achieved（ 39. 05 ± 1. 19） %. When the transfected ratio was fixed,transfection efficiency increased first and then decreased with the increase of the amount of plasmid. When the ratio of FuGENE and plasmid was 5 μL∶ 2. 5 μg,the highest transfection efficiency was achieved（ 42. 98 ± 2. 56） %. The results of qRT-PCR showed that the mRNA levels of HSF1 in the transfected group were significantly higher than those in the control group.Conclusion： For JEG-3 cells,the best transfection ratio was 5 μL∶ 2. 5 μg by using Fugene reagent.The result laid the experimental foundation for the research of gene HSF1.

关 键 词：热休克转录因子 氧化应激 绿色荧光蛋白 人绒毛膜癌细胞 转染 DNA延滞实验 质粒 FuGENE 

分 类 号：R-33[医药卫生] R363