Therapeutic ultrasound promotes plasmid DNA uptake by clathrin-mediated endocytosis
Therapeutic ultrasound promotes plasmid DNA uptake by clathrin-mediated endocytosis
复制标题
DOI：
10.1002/jgm.1586
复制
发表时间：
2011-07-01
影响因子：
3.5
通讯作者：
Han, Sang Won
中科院分区：
文献类型：
--
作者：
Bentes de Paula, Daisy Maria;Valero-Lapchik, Valderez Bastos;Han, Sang Won
背景超声已被广泛应用于提高非病毒载体转染效率。质粒摄取的机制通常归因于声致穿孔，尽管这种归因没有明确的证据。根据我们之前的结果，我们假设其他机制（例如内吞作用）可能参与了这一过程。方法使用无微泡的治疗性超声将质粒载体pEGFP-N3（4.7kb）转染NIH 3 T3细胞。生物效应，如钙离子内流，活性氧（ROS）的产生和膜电位的改变，通过共聚焦显微镜实时超声辐照后的荧光染料。结果超声功率为2 W/cm（2），占空比为20%，持续30 s，转染效率约为40%，但功率为1 W/cm（2）时，转染效率很低。在超声波照射期间，ROS和钙内流的产生都增加了，尽管它们在关闭US后不久就停止了，但钙内流以1 W/cm（2）除外。超声还使细胞膜电位向超极化状态转变，并在照射后不久恢复正常。标记的质粒DNA与网格蛋白介导的内吞标记物共定位，但与小窝蛋白-1不共定位。结论超声促进质粒DNA的摄取可能是通过网格蛋白介导的内吞作用进行的。版权所有（C）2011约翰威利父子有限公司
Background Ultrasound (US) has been widely used to improve the efficiency of nonviral vector transfection. The mechanism of plasmid uptake is usually attributed to sonoporation, although there is not clear evidence for this attribution. Based on our previous results, we hypothesized that other mechanisms, such as endocytosis, could be involved in this process.Methods NIH3T3 cells were transfected with plasmid vector pEGFP-N3 (4.7kb) using a therapeutic US without microbubbles. Bioeffects such as calcium influx, reactive oxygen species (ROS) generation and membrane potential alterations were accessed with fluorescent dyes in real-time by confocal microscopy after US insonation. Localization of labeled plasmid DNA in cells was also monitored with endocytosis markers using an immunofluorescence assay.Results US at 2 W/cm(2) with a duty-cycle of 20% for 30 s resulted in approximately 40% transfection efficiency but, at 1 W/cm(2), resulted in a very low level of transfection. Both the production of ROS and calcium influx were augmented during the insonation, although they were stopped soon after turning off US, with the exception of calcium influx with 1 W/cm(2). US also changed the cell membrane potential to the hyperpolarization state, which returned to the normal state soon after insonation. Labeled plasmids DNA could be co-localized with clathrin-mediated endocytosis marker but not with caveolin-1.Conclusions The present data indicate that plasmid DNA uptake promoted by US should occur via clathrin-mediated endocytosis. Copyright (C) 2011 John Wiley & Sons, Ltd.