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.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2009年第24期2459-2462,共4页
Journal of Third Military Medical University
关键词
超声学
造影剂
人牙龈成纤维细胞
基因转染
utrasonics
contrast media
human gingival fibroblasts
gene transfection
