Gene transfer to corneal epithelium and keratocytes mediated by ultrasound with microbubbles
Gene transfer to corneal epithelium and keratocytes mediated by ultrasound with microbubbles
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DOI：
10.1167/iovs.05-0889
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发表时间：
2006-02-01
影响因子：
4.4
通讯作者：
Sakamoto, T
中科院分区：
文献类型：
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作者：
Sonoda, S;Tachibana, K;Sakamoto, T
目的.角膜是评估基因转移的理想器官，因为它可以进行非侵入性治疗并易于监测。本研究旨在探讨超声（US）联合微泡（MB）介导的角膜基因转移的有效性和安全性。将培养的兔角膜上皮（RC-1）细胞在24孔培养皿中与具有在巨细胞病毒启动子下的绿色荧光蛋白（GFP）基因的质粒DNA一起孵育。将细胞暴露于不同强度的US（1 MHz;功率，0.5类似于2 W/cm（2）;持续时间，15 - 120秒;占空比，20% - 100%）。还检查了用MB同时刺激的效果。通过在显微镜下计数GFP阳性细胞的数量来定量基因转移。此外，通过将GFP质粒注射到兔角膜中和用MBs. pETS. US暴露来检查体内基因转移。在体外研究中，单独的DNA暴露无法将基因转移到培养的RC-1细胞中; US略微增强了基因转移。与MBs共暴露显著增加基因转移效率。在体内研究中，单独注射DNA可以在有限的程度上转移基因，但质粒注射+超声与MB的联合使用可以显著提高基因转移效率，且无明显的组织损伤，实现了基因的二维转移。超声与微球大大增加了体内和体外角膜细胞的基因转移。这种非侵入性的基因转移方法可能是一种有用的工具，临床基因治疗。
PURPOSE. The cornea is an ideal organ for evaluating gene transfer because it can be treated noninvasively and monitored easily. The present study was performed to investigate the practical efficacy and safety of ultrasound ( US) plus microbubble (MB)-mediated gene transfer to cornea.METHODS. Cultured rabbit corneal epithelial (RC-1) cells were incubated in 24-well dishes with plasmid DNA having a green fluorescent protein (GFP) gene under a cytomegalovirus promoter. The cells were exposed to US under different intensities (1 MHz; power, 0.5 similar to 2W/cm(2); duration, 15 - 120 seconds; duty cycle, 20% - 100%). The effect of simultaneous stimulation with MBs was also examined. Gene transfer was quantified by counting the number of GFP-positive cells under microscopy. Furthermore, in vivo gene transfer was examined by GFP plasmid injection into rabbit cornea and US exposure with MBs.RESULTS. In the in vitro study, DNA exposure alone could not transfer gene into cultured RC-1 cells; US enhanced gene transfer slightly. Coexposure with MBs significantly increased gene transfer efficiency. In the in vivo study, DNA injection alone could transfer the gene to a limited degree, but plasmid injection plus US with MBs strongly increased gene transfer efficiency without apparent tissue damage, and gene transfer was achieved two dimensionally.CONCLUSIONS. US with MBs greatly increases gene transfer to in vivo and in vitro corneal cells. This noninvasive gene transfer method may be a useful tool for clinical gene therapy.