超声微泡造影剂介导pEGFP-N_1质粒转染HGFs的实验研究  

Ultrasound-mediated microbubble destruction enhances GFP gene expression in human gingival fibroblasts cells in vitro

作　　者：骆书美[1] 张云燕[1] 钟晓波[1] 仲琳[1] 

机构地区：[1]重庆医科大学附属口腔医院牙体牙髓科,重庆400015

出　　处：《第三军医大学学报》2009年第24期2459-2462,共4页Journal of Third Military Medical University

摘　　要：目的探讨超声破坏微泡介导pEGFP-N1质粒转染人牙龈成纤维细胞的可行性、优化转染的条件及转染效率。方法体外原代培养HGFs。以pEGFP-N1质粒为报告基因,微泡造影剂为载体,用超声辐照介导质粒pEGFP-N1转染HGFs。实验组分为质粒组、微泡+质粒组、超声+质粒组、超声+微泡+质粒组,超声+微泡+质粒组按不同的转染条件分成亚组。转染48h后倒置荧光显微镜下观察GFP的表达,MTT法检测HGFs活性。结果超声微泡介导pEGFP-N1质粒对HGFs的转染效率明显高于其他实验组(P<0.05)。优化转染条件后,频率300KHz,声强1.5W/cm2,连续波,辐照时间60s,微泡浓度10%,质粒浓度6.67μg/ml时,转染率较高。结论在一定条件下,超声微泡造影剂能够促进外源基因对HGFs的转染。Objective To investigate whether ultrasound-mediated microbubble destruction can effectively deliver pEGFP-N1 plasmid into human gingival fibroblasts （HGFs）. Methods The primary cultured HGFs were divided into 4 groups, that is, plasmid, mierobubble ＋ plasmid, ultrasound ＋ plasmid, and ultrasound ＋ microbubble ＋ plasmid groups, pEGFP-N1 plasmid was used to transfect to HGFs as a gene marker with ultrasound-mediated microbubble destruction. The last group was further divided into subgroups to optimize the transfection conditions. After 48 h, phase-contrast fluorescent microscopy was employed to evaluate the expression of green fluorescent protein （GFP）. The cell vitality was measured by the MTF assay. Results The transfection efficiency of the ultrasound ＋ microbubble ＋ plasmid group was higher than other experiment groups. Optimal gene expression was found when ultrasound was radiated at 1.5 W/cm^2 for 60 s, microbubble was at a concentration of 10% and plasmid was at a concentration of 6.67 μg/ml, and the transfection efficiency was highest under this condition. Conclusion Under specific conditions, ultrasound mediated microbubble destruction enhances the reporter gene transfeetion and expression in HGFs.

关 键 词：超声学 造影剂 人牙龈成纤维细胞 基因转染 

分 类 号：R322.41[医药卫生—人体解剖和组织胚胎学] R394-33[医药卫生—基础医学]