摘要
目的探索超声辐照微泡介导腺相关病毒（AAV）对不同细胞的转染和细胞内吞活动。方法以不同梯度浓度的携增强型绿色荧光基因的重组腺相关病毒2型（rAAV2-EGFP）转染人宫颈癌细胞（HeLa）和小鼠成纤维细胞（NIH／3T3）细胞，分别获得最佳可提高基础浓度。以此浓度，超声辐照微泡介导rAAV2-EGFP转染HeLa和NIH／3T3细胞后48h荧光显微镜观察和流式细胞仪检测EGFP基因转染效率，并采用CCK-8检测其安全性。超声辐照微泡介导AAV转染后45min，采用免疫荧光的方法共聚焦显微镜和透射电镜观察细胞膜内网格蛋白包裹的内吞泡的数量和分布。结果rAAV2-EGFP转染HeLa和NIH／3T3细胞的最佳可提高浓度分别为2000v．g．／cell和10000v．g．／cell。超声辐照微泡可显著提高rAAV2-EGFP对HeLa和NIH／3T3细胞的转染效率（P〈0．01），而细胞活性无显著下降（P〉0．05）。共聚焦显微镜和电镜显示，超声辐照微泡介导后，细胞膜内侧面网格蛋白包裹的内吞泡明显增多。结论超声辐照微泡可提高有赖于细胞转运的AAV病毒颗粒对两种不同细胞的基因转染，激活细胞内吞活动可能是超声辐照微泡促进AAV转运的机制之一。
Objective To explore the adeno-associated virus （AAV） gene transduction and cellular endocytosis mediated by ultrasound combined with microbubbles in two types of cells. Methods HeLa and NIH/3T3 cells were infected by rAAV2-EGFP at a concentration gradient to get the optimal concentrations for enhancement. At these concentrations, HeLa and NIH/3T3 cells were infected by rAAV2-EGFP mediated by ultrasound combined with microbubbles. The gene transduction efficiency were observed and measured by fluorescence microscopy and flow cytometry at 48 h after treatment. The cell viability was tested by CCK-8. The number and distribution of cellular c[athrin-coated endoeytic pits were observed by confocal fluorescence microscopy and transmission electron microscopy on 45 min after treatment. Results The optimal concentrations for HeLa and NIH/3T3 cells were 2000 v.g./cell and 10000 v.g./cell.Ultrasound combined with microbubbles significantly enhanced the transduetion efficiency of rAAV2-EGFP （ P 〈0.01） without significant cell viability decrease （P 〉0.05）. Confocal fluorescence microscopy and transmission electron microscopy demonstrated that elathrin-eoated endocytic pits were more obviously increased in ultrasound combined with microbubbles mediated AAV transduction group than AAV transduction group. Conclusions Ultrasound combined with microbubbles can efficiently enhance the gene transduction of AAV,whose cellular transportation depends on cellular endocytosis, in two types of cells. Stimulating cellular endocytosis might be one of the mechanisms of enhanced cellular transportation of AAV mediated by ultrasound combined with microbubbles.
出处
《中华超声影像学杂志》
CSCD
北大核心
2015年第9期809-813,共5页
Chinese Journal of Ultrasonography
基金
国家自然科学基金（81401415,81471666）
上海交通大学“医工交叉研究基金”（YG2013MS02）
关键词
超声处理
微气泡
腺相关病毒
转染
细胞内吞
Sonication
Microbubbles
Adeno-associated virus
Transfection
Cellular endocytosis
