优化脂质体对肝癌细胞株HepG2细胞转染效率

Optimization of Transfection Efficiency in Hepatocellular Carcinoma HepG2 Cells with Lipofectamine

摸索用Lipofectamine 2000(Lipo)转染质粒pEGFP-C1到肝癌细胞HepG2较为合适的转染条件。以HepG2细胞为研究对象,采用脂质体Lipofectamine 2000转染pEGFP-C1质粒,在24孔板先按每孔0.5μg固定pEGFP-C1质粒用量,摸索Lipo在1μL、1.5μL、2μL、2.5μL量上较为合适的用量,确定Lipo用量后,然后固定Lipo为1μL,摸索质粒用量0.5μg、1μg,确定脂质体与质粒的最佳比例。此外,对转染中培养基是否含血清,Lipofectamine 2000与pEGFP-C1质粒比例为1:0.5的基础上,扩大用量,即Lipofectamine 2000 2μL和pEGFP-C1质粒1μg,以及脂质体复合物孵育细胞时间进行优化。最后,采用荧光倒置显微镜观察细胞转染效率和方差分析及秩和检验的统计学方法进行统计分析。结果显示,pEGFP-C1质粒固定为0.5μg时,Lipo用量在1μL及1.5μL用量组转染效率最好,之后随着Lipo用量加大,转染效率下降;固定Lipo用量1μL,pEGFP-C1质粒0.5μg时转染效率最好(p<0.05),比例不变,扩大Lipo和质粒用量,并不增加转染效率。转染后于3 h、6 h、8 h、12 h、24 h换成正常培养基培养,转染6 h后换液较好。此外,研究发现培养基是否含血清不影响转染效率。本研究表明在24孔板板中用Lipofectamine 2000转染HepG2细胞时较为合适的转染条件为每孔1μL Lipofectamine 2000和0.5μg质粒,脂质体与质粒的最佳比例为2:1,血清不影响转染效率,用含血清培养基转染后6 h换液培养。

Abstract To explore the suitable transfection conditions of transfecting pEGFP-C1 plasmid into hepatocellular carcinoma HepG2 cells by Lipofectamine 2000, in this sttudy, HepG2 cells was used as the research objects and pEGFP-C 1 was introduced into HepG2 cells with Lipofectamine 2000. Firstly, the amount of pEGFP-C1 plasmid was fixed to 0.5 pug per well in 24-well plate, and then different amounts of Lipofectamine 2000 （1μL, 1.5 μL, 2 μL, 2.5 μL） was added respectively. Secondly, after determining the amount of lipo is 1μL, plasmid DNA pEGFP-C1 was added to 1μL of Lipofectamine 2000 at different amounts ofpEGFP-C1 （0.5μg, 1 μg） respectively. At the same time mediun containing serum and serum free was compared during transfection. Based on the result of ratio of Lipofectamine 2000 to pEGFP-C1 was 1： 0.5, the amount of Lipofectamine 2000 and pEGFP-C1 were enlarged to 2 μL and 1 μg, respectively. Additionally, incubation time of cell with Lipofectamine complex was optimized. Finally, fluorescence microscope was used to observe the transfection efficienc, analysis of variance and rank sum test were used to do statistics analysis. The results showed that when the amount of pEGFP-C1 was 0.5 μg, the transfection efficiency was the best when the amount of Lipofectamine 2000 were 1 μL and 1.5 μL. The transfection efficiency was decreased along with increase of the amount of Lipofectamine 2000. Forthermore, when the amount of Lipofeetamine 2000 was fixed to 1 μL, the transfection efficiency of 0.5 μg pEGFP-CI was significantly higher than that of 1μg （p〈0.05）. The group of 1 μL Lipofectamine 2000 and 0.5μg pEGFP-CI was significantly higher than 2 μL Lipofectamine 2000 and 1μLpEGFP-C 1 （p〈0.05）, expanding amount did not improve transfection efficiency. After transfection, the normal medium was replaced in 3 h, 6 h, 8 h, 12 h, 24 h. The better incubation time of cell with liposome complex is 6 h after transfecting with normal medium. There was no statistical significance compared transfection media cotaining serum or no serum （p〉0.05）. This study indicated that the suitable transfection conditions in 24 well plate are 1μL Lipofectamine 2000 and 0.5μg pEGFP-CI, the ratio of Lipofectamine and plasmid is 2： 1, serum do not affect the efficiency of transfection. Moreover, medium was replaced after incubation liposome complex for 6 hours.