三种试剂转染293T细胞的效率及毒性比较  

Comparative Study of the Transfection Efficiency and Toxicity of 293T Cells Transfected by Three Reagents

作　　者：陈凤 靳兴汉 王菲[1] CHEN Feng;JIN Xinghan;WANG Fei(Digestive Medicine Center,the Seventh Affiliated Hospital,Sun Yat-sen University,Shenzhen 518107,China)

机构地区：[1]中山大学附属第七医院,消化医学中心,深圳518107

出　　处：《中国细胞生物学学报》2021年第8期1630-1637,共8页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:81800525、81873573)资助的课题。

摘　　要：该文旨在比较不同的转染试剂介导两种荧光素酶报告基因转染人胚肾细胞293T的效果。采用脂质体转染试剂Lipofectamine?2000、非脂质体成分X-tremeGENE HP DNA和FuGENE?HD转染试剂,选取不同配比的质粒DNA和转染试剂分别将含有绿色荧光蛋白(GFP)和红色荧光蛋白(RFP)的质粒转入293T细胞中,应用倒置荧光显微镜和流式细胞仪分析转染效率,锥虫蓝染色法检测细胞存活率。X-tremeGENE HP DNA转染试剂转染293T细胞的效率最高,不管目的基因、质粒DNA和转染试剂的配比如何,其转染效率均显著高于脂质体转染试剂(P均<0.05);在相同的质粒DNA和转染试剂比例下,FuGENE?HD试剂介导GFP转染的效率显著高于脂质体试剂Lipofectamine?2000(P均<0.05),但二者介导RFP转染的效率无显著性差异(P均>0.05);在质粒DNA和转染试剂比为1?2时,X-tremeGENE HP DNA试剂转染GFP和RFP的效率均显著高于FuGENE?HD试剂(P均<0.05),当比例递变为1?4时,二者的转染效率无显著性差异(P均>0.05)。随着转染试剂使用量的增多,Lipofectamine?2000和FuGENE?HD试剂的转染效率明显升高,而X-tremeGENE HP DNA试剂的转染效率则下降。与转染GFP基因相比,三种试剂的转染效率均随着目的基因RFP片段的增大而显著降低(P均<0.05)。在质粒DNA和转染试剂的比例相同的情况下,三种试剂转染后的存活率无显著性差异(P均>0.05),但随着转染试剂用量的增加,三种试剂转染后的细胞存活率均显著降低(P均<0.05)。目的基因大小与转染效率成反比,综合考虑转染试剂用量、转染效率和细胞活性,该研究认为X-tremeGENE HP DNA转染试剂效果最好。This paper aims to compare the transfection efficiencies of two luciferase reporter genes into human embryonic kidney cells 293T by different transfection reagents.By using Lipofectamine?2000,X-tremeGENE HP DNA,FuGENE?HD transfection reagents that with different ratios of plasmids DNA and transfection reagents,the plasmids containing GFP(green fluorescent protein)and RFP(red fluorescent protein)were transfected into 293T cells,respectively.The transfection efficiency was analyzed by inverted fluorescence microscope and flow cytometry,and the cell survival rate was detected by trypan blue stain.Regardless of the target gene and ratio of plasmid DNA to transfection reagents,the transfection efficiency of X-tremeGENE HP DNA was significantly higher than Lipofectamine?2000(all P<0.05).Under the same ratio of plasmid DNA to transfection reagent,the efficiency of FuGENE?HD mediated GFP was significantly higher than Lipofectamine?2000(all P<0.05),but there was no significant difference of RFP transfection between the two reagents(all P>0.05).When the ratio of plasmid DNA to transfection reagent was 1:2,the efficiency of X-tremeGENE HP DNA mediated GFP and RFP transfection was significantly higher than FuGENE?HD reagent(all P<0.05),When the ratio of plasmid DNA to transfection reagent was changed to 1:4,the transfection efficiency between the two reagents was not significantly different(all P>0.05).With the increase of the amount of transfection reagents,the efficiencies of Lipofectamine?2000 and FuGENE?HD transfection reagent were increased significantly,whereas the transfection efficiency of X-tremeGENE HP DNA transfection reagent was decreased significantly.Compared with GFP gene transfection,the transfection efficiency was significantly decreased with the increase of target gene RFP fragment(all P<0.05).When the ratio of plasmid DNA to transfection reagent was the same,there was no significant difference in the survival rate after transfection among the three reagents(all P>0.05),while when the amount of transfection

关 键 词：转染试剂 293T 转染效率 细胞活性 

分 类 号：Q78[生物学—分子生物学]