DNA delivery to 'ex vivo' human liver segments
DNA delivery to 'ex vivo' human liver segments
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DOI：
10.1038/gt.2011.144
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发表时间：
2012-05-01
期刊：
影响因子：
5.1
通讯作者：
Alino, S. F.
中科院分区：
文献类型：
--
作者：
Herrero, M. J.;Sabater, L.;Alino, S. F.
流体动力学注射是一种有效的程序，在啮齿类动物的肝脏基因治疗，但在大型动物的疗效有限，使用“在体内”适应区域流体动力学基因传递系统。我们研究的能力，这一程序介导的基因传递在人类肝段获得手术切除。在不同的流速（1、10和20 ml/s）和最终灌注体积条件下，将含有增强型绿色荧光蛋白（eGFP）基因质粒的盐水溶液从肝静脉逆行注射到水密性肝段。将样品培养1至2天，并用于基因表达的显微镜和分子分析。荧光和免疫组织化学研究表明，在注射>= 10 ml/s（-1）的节段中，肝脏切片中存在良好和广泛的基因表达，分子分析以定量方式加强了组织学观察（表观基因递送指数：10（2）-10（4）eGFP DNA拷贝/100 pg总DNA;转录指数：10（5）-2 × 10（6）eGFP RNA拷贝/100 ng总RNA）。此外，注射的金纳米颗粒（直径15纳米）表明，DNA向肝细胞的递送必须涉及促进渗透过程，而不会破坏膜。总之，我们表明，水密性人肝段的逆行静脉注射是一种溯河产卵程序，导致广泛的肝脏基因递送和良好的基因表达。然而，在我们的模型中，需要进一步的研究来阐明长期缺血损伤对肝细胞的影响。Gene Therapy（2012）19，504-512; doi：10.1038/gt.2011.144; 2011年10月13日在线发表
Hydrodynamic injection is an efficient procedure for liver gene therapy in rodents but with limited efficacy in large animals, using an 'in vivo' adapted regional hydrodynamic gene delivery system. We study the ability of this procedure to mediate gene delivery in human liver segments obtained by surgical resection. Watertight liver segments were retrogradely injected from hepatic vein with a saline solution containing a plasmid bearing the enhanced green fluorescent protein (eGFP) gene, under different conditions of flow rate (1, 10 and 20 ml s(-1)) and final perfused volume. Samples were cultured for 1 to 2 days and used for microscopy and molecular analysis of gene expression. The fluorescent and immunohistochemistry studies indicated that in segments injected at >= 10 ml s(-1), good and wide gene expression was present in the liver sections and the molecular analysis reinforced the histological observation in a quantitative manner (index of apparent gene delivery: 10(2)-10(4) eGFP DNA copy per 100 pg of total DNA; transcription index: 10(5)-2x10(6) eGFP RNA copy per 100 ng of total RNA). In addition, injected gold nanoparticles (15 nm diameter) suggested that DNA delivery to hepatocytes must involve a facilitated permeation process without membrane disruption. In summary, we show that retrograde venous injection of watertight human liver segment is an anadromous procedure that results in wide liver gene delivery and good gene expression. However, additional studies will be necessary to clarify the influence of the prolonged ischemia injury to hepatocytes in our model. Gene Therapy (2012) 19, 504-512; doi: 10.1038/gt.2011.144; published online 13 October 2011