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冲击波与DNA-阳离子脂质组装体:一种在人类细胞中表达外源基因的协同方法。

Shock waves and DNA-cationic lipid assemblies: a synergistic approach to express exogenous genes in human cells.

作者信息

Millán-Chiu Blanca, Camacho Giselle, Varela-Echavarría Alfredo, Tamariz Elisa, Fernández Francisco, López-Marín Luz M, Loske Achim M

机构信息

Centro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro, México.

Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro, México.

出版信息

Ultrasound Med Biol. 2014 Jul;40(7):1599-608. doi: 10.1016/j.ultrasmedbio.2014.01.012. Epub 2014 Mar 15.

Abstract

Cationic lipid/DNA complexes (lipoplexes) represent a powerful tool for cell transfection; however, their use is still limited by important concerns, including toxicity and poor internalization into deep tissues. In this work, we investigated the use of shock wave-induced acoustic cavitation in vitro for the transfection of lipoplexes in human embryo kidney 293 cells. We selected shock waves with the ability to internalize 10-kDa fluorescein isothiocyanate-dextran into cells while maintaining survival rates above 50%. Cell transfection was tested using the green fluorescent protein-encoding plasmid pCX::GFPGPI2. Confocal microscopy and fluorescence-assisted cell sorting analyses revealed successful transfection after treatments ranging from 1 to 3 min using 60 to 180 shock waves at peak amplitudes of 12.3 ± 1.5 MPa. Interestingly, the combination of shock waves and lipoplexes induced a 3.1- and 3.8-fold increase in the expression of the reporter gene compared with the use of lipoplexes or shock waves alone, respectively. These results indicate that cationic DNA assembly and shock waves act in a synergistic manner to promote transfection of human cells, revealing a potential approach for non-invasive site-specific gene therapy.

摘要

阳离子脂质/DNA复合物(脂质体复合物)是细胞转染的有力工具;然而,它们的应用仍受到一些重要问题的限制,包括毒性以及难以有效内化进入深部组织。在本研究中,我们在体外研究了冲击波诱导的声空化作用对人胚肾293细胞中脂质体复合物转染的影响。我们选择了能够将10 kDa异硫氰酸荧光素 - 葡聚糖内化进入细胞,同时保持细胞存活率高于50%的冲击波。使用编码绿色荧光蛋白的质粒pCX::GFPGPI2测试细胞转染情况。共聚焦显微镜和荧光辅助细胞分选分析表明,在峰值振幅为12.3±1.5 MPa的条件下,使用60至180次冲击波处理1至3分钟后,成功实现了转染。有趣的是,与单独使用脂质体复合物或冲击波相比,冲击波和脂质体复合物的组合分别使报告基因的表达增加了3.1倍和3.8倍。这些结果表明,阳离子DNA组装和冲击波以协同方式促进人类细胞的转染,揭示了一种非侵入性位点特异性基因治疗的潜在方法。

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