摘要
旨在分析生长抑素重组质粒pEGS/2SS转染HeLa细胞(人宫颈癌上皮细胞)转染效果的影响因素,探讨最佳转染条件,为进一步研究生长抑素基因疫苗的作用机制和作用效果奠定基础。以脂质体转染法将带有绿色荧光蛋白(GFP)报告基因的重组质粒pEGS/2SS转染HeLa细胞,探讨质粒转染HeLa细胞最佳条件的4个参数:质粒DNA剂量、脂质体剂量、最佳转染时间和质粒提取方法,在荧光显微镜下观察细胞转染情况并计算转染率。结果:在六孔细胞培养板上,当DNA/脂质体剂量为1μg/6μg时,转染效率最高,在转染后72 h达到34.02%;当用Plasmid Maxi Kit试剂盒提取质粒,转染时间为48 h,转染效率最高,达到17.88%。重组质粒pEGS/2SS转染He-La细胞条件是:采用试剂盒(Plasmid Maxi Kit)提取的质粒,DNA和脂质体的剂量分别为1μg与6μg,转染时间48 h,此时的转染效率最高,达17.88%。
We aimed to investigate the factors infecting transfection efficiency of somatostatin recombinant plasmid pEGS/2SS introducting into HeLa cells, make preparations for advanced study on somatostatin gene vaccine mechanism and effect of action. Four parameters including transfeetion reagent （lipofectamineTM） concentration, DNA concentration, cells cultured time after transfection and DNA extracting techniques were analyzed and optimized, and transfeetion effi- ciency was evaluated by the percentage of transfected cells with green fluorescence under fluorescence microscope. Based on the optimized lipofectamineTM and DNA concentration, 34. 02% transfected HeLa cells could express green fluorescence 72 h after transfection when the concentration of lipofeetamineTM and DNA was individually 6μg and 1μg; Based on the optimized cells cultured time after transfection and DNA extracting techniques, 17.88% transfeeted HeLa cells could express green fluorescence protein with Plasmid Maxi Kit extracting DNA 48 h after trasfection. Optimal transfection conditions were determined with four parameters, 17.88% HeLa cells could express green fluorescence protein with Plasmid Maxi Kit extracting DNA, 1μg DNA and 6μg lipofectamineTM 48 h after transfection. K
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2010年第3期279-285,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(30771549)
