pEGFP-N1脂质体转染方法的优化及其应用

目的: 应用脂质体介导的转染方法将pEGFP-N1转染入Fisher大鼠甲状腺滤泡上皮细胞中,并获得较佳的优化方案,阐明该优化条件在转染其他目的基因时的适用性。方法: 分别选取不同配比的质粒和脂质体,将pEGFP-N1转入Fisher大鼠甲状腺滤泡上皮细胞。转染后36h,倒置荧光显微镜和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;台盼蓝染色法检测细胞存活率,以此获得优化的转染条件。同时将pEGFP-Aquaporin2、pEGFP-Aquaporin4和pEGFP-Aquaporin5分别以上述条件转染,并获取相应优化的转染条件。结果: 在质粒质量固定的条件下,随着脂质体质量的增加,其转染效率和细胞存活率升高;但是当质粒:脂质体>1:4时,转染效率和细胞存活率下降。在质粒和脂质体比例固定而质量增加时,其转染效率和细胞存活率下降;当质粒:脂质体>3.2μg:12.8μL时,转染效率降低。应用脂质体介导的方法转染pEGFP-N1和转染pEGFP-Aquaporin2、pEGFP-Aquaporin4和pEGFP-Aquaporin5时,在质粒与脂质体的配比为1:4(3.2μg:12.8μL)时,转染效率达最高。结论: 在应用脂质体介导的目的基因转染时,pEGFP-N1优化的转染条件具有较为广泛的适用性,可用作后续目的基因高效转染优化条件。

Optimization of liposome-mediated transfection of pEGFP-N1 and its applification

Objective: To optimize the liposome-mediated transfection conditions based on transfecting the pEGFP-N1 into thyroid cells of Fischer rats,and to investigate its effectiveness on other target gene transfection. Methods: Based on the different ratios and quantities of plasmids and liposomes,the thyroid follicular epithelial cells of Fischer rats were transfected with pEGFP-N1.The expression of enhanced green fluorescence protein(EGFP) and transfection efficiency were observed under inverted fluorescence microscope and flow cytometry, respectively.Then the cell viability was detected by Trypan Blue stainning.Meanwhile the thyroid follicular epithelial cells of Fischer rats were transfected with pEGFP-Aquaporin2,pEGFP-Aquaporin4 and pEGFP-Aquaporin5 under the above conditions,and the optimized conditions of transfecion were obtained. Results: When the quantity of plasmid was certain,the transfection efficiency and cell viability were increased with the increasing of liposomes,but the transfection efficiency and cell viability were decreased when the ratio of plasmid to liposome was over 1:4.When the ratio of plasmid to liposome was certain and the quantities were increased gradully,the transfection effciency was increased,and the cell viability was decreased.With the ratio of plasmids to liposomes was 1:4 (3.2 μg:12.8 μL),the transfecion efficencies of pEGFP-N1,pEGFP-Aquaporin2,pEGFP-Aquaporin2, and pEGFP-Aquaporin2 reached the highest values,respectively. Conclusion: The optimized conditions of transfection pEGFP-N1 is the same as the other plasmids, which can be used as optimized conditions for efficient transfection of the other target gene.