Ex vivo Gene Electrotransfer to the Endothelium of Organ Cultured Human Corneas
Ex vivo Gene Electrotransfer to the Endothelium of Organ Cultured Human Corneas
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DOI：
10.1159/000246577
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发表时间：
2010-01-01
影响因子：
2.1
通讯作者：
Thuret, Gilles
中科院分区：
文献类型：
--
作者：
He, Zhiguo;Pipparelli, Aurelien;Thuret, Gilles
目的：描述一种创新的装置，允许基因电转移到人角膜内皮细胞（EC）在器官培养储存期间。方法：开发无内皮接触的定制电极。将含有巨细胞病毒启动子和报告基因[增强型绿色荧光蛋白（eGFP）或β-半乳糖苷酶（β-gal）]的两种质粒用8个125 mA方波电流的1 Hz 100 ms脉冲电穿孔到2组人角膜中。对照暴露于无电脉冲的裸DNA。eGFP转导的角膜用于确定转基因表达动力学，而β-gal使用图像分析工具测量转染效率。总的来说，内皮毒性通过以下测定：（1）在电穿孔后3小时和3天以及14天，在15个eGFP转染的成对角膜上使用Hoechst 33342、乙锭同二聚体III和钙黄绿素AM的三重染色的细胞毒性测试;（2）抗ZO-1染色以评估紧密连接的完整性。结果：所有电穿孔的角膜携带转染的EC，而对照不携带。在电转移后3小时观察到eGFP表达，然后从第1天到第28天存在。在63个用β-gal转染的角膜上测定的转染效率范围为转染EC的0.1%至54%（平均值+/- SD：7 +/-11%，中值：2.9%），对于来自相同供体的配对角膜具有显著的再现性。电穿孔产生低的早期EC死亡。抗ZO-1染色显示EC镶嵌连续性没有显着变化，无论是1和3，也不是28天后电穿孔。结论：用定制设计的电极将基因电转移到器官培养的人角膜的内皮，允许快速和容易的EC转染。然而，需要进一步优化以确保可重现的结果。版权所有（C）2009 S. Karger AG，巴塞尔
Aims: To describe an innovative device that allows gene electrotransfer to human corneal endothelial cells (EC) during storage in organ culture. Methods: Customized electrodes without endothelial contact were developed. Two plasmids containing the cytomegalovirus promoter and reporter genes [enhanced green fluorescent protein (eGFP) or beta-galactosidase (beta-gal)] were electroporated in 2 series of human corneas with eight 1-Hz 100-ms pulses of 125 mA square current. Controls were exposed to naked DNA without electric pulses. eGFP-transduced corneas were used to determine the transgene expression kinetics, whereas beta-gal measured transfection efficiency using image analysis tools. Overall, endothelial toxicity was determined by: (1) cytotoxicity tests using triple staining with Hoechst 33342, ethidium homodimer III, and calcein AM, 3 h and 3 and 14 days after electroporation on the series of 15 eGFP-transfected paired corneas; (2) anti-ZO-1 staining to assess tight junctions' integrity. Results: All electroporated corneas carried transfected ECs, whereas the controls carried none. eGFP expression was observed 3 h after electrotransfer, and was then present from days 1 to 28. Transfection efficiency determined on 63 corneas transfected with beta-gal ranged from 0.1 to 54% of the transfected ECs (mean +/- SD: 7 +/- 11%, median: 2.9%) with significant reproducibility for paired corneas from the same donor. Electroporation produced low early EC death. Anti ZO-1 staining revealed no dramatic change in EC mosaic continuity, neither 1 and 3 nor 28 days after electroporation. Conclusions: Gene electrotransfer to the endothelium of organ-cultured human corneas with custom-designed electrodes allows rapid and easy EC transfection. However, further optimization is required to ensure reproducible results. Copyright (C) 2009 S. Karger AG, Basel