Hellgren I, Drvota V, Pieper R, Enoksson S, Blomberg P, Islam K B, Sylvén C
Department of Cardiology, The Clinical Research Center, Huddinge, Stockholm, Sweden.
Cell Mol Life Sci. 2000 Aug;57(8-9):1326-33. doi: 10.1007/pl00000769.
The present study was undertaken to develop an efficient non-viral gene delivery system for cardiovascular gene therapy. We investigated transfection efficiency and toxic properties of the new transfection reagent, FuGene6, and compared it with two other transfection reagents, Tfx-50 and LipoTaxi. For in vivo experiments, the plasmid was delivered intramuscularly via transplantation of fibroblasts transfected with plasmid and FuGene6. Conditions for efficient gene delivery were initially studied in vitro. Human and rabbit fibroblasts were isolated from skin, cultured and transfected with phVEGF165 or pCMVbeta gal plasmids, coding for vascular endothelial growth factor (VEGF) or beta-galactosidase, respectively. The effect of the DNA amount and the DNA:transfection reagent ratio on plasmid uptake were studied. Of the transfection reagents tested, only FuGene6 provided high-efficiency and dose-dependent plasmid transfer both for cell-localised (beta-galactosidase) and secreted (VEGF) gene products. When analysed with an MTT assay, FuGene6 showed no toxicity at low doses. Optimised conditions were applied for in vivo reporter gene delivery. Rabbits were injected intramuscularly with ex vivo-transfected fibroblasts. As in in vitro studies, ex vivo-transfected fibroblasts showed highly efficient gene expression in vivo. Tissue sections were analysed with macrophage-specific immunostaining. No signs of inflammation were seen in the region of fibroblast injection. This study demonstrates that FuGene6 is a highly efficient transfection reagent that may be useful for in vitro non-viral transfection of primary human and rabbit fibroblasts and for in vivo therapeutic non-viral gene delivery.
本研究旨在开发一种用于心血管基因治疗的高效非病毒基因递送系统。我们研究了新型转染试剂FuGene6的转染效率和毒性特性，并将其与另外两种转染试剂Tfx - 50和LipoTaxi进行比较。对于体内实验，通过移植用质粒和FuGene6转染的成纤维细胞，将质粒肌肉注射递送。高效基因递送的条件首先在体外进行研究。从皮肤中分离出人及兔的成纤维细胞，进行培养，并用分别编码血管内皮生长因子（VEGF）或β - 半乳糖苷酶的phVEGF165或pCMVbeta gal质粒进行转染。研究了DNA量和DNA与转染试剂比例对质粒摄取的影响。在所测试的转染试剂中，只有FuGene6对于细胞定位（β - 半乳糖苷酶）和分泌（VEGF）基因产物都提供了高效且剂量依赖性的质粒转移。用MTT法分析时，FuGene6在低剂量下无毒性。将优化条件应用于体内报告基因递送。给兔子肌肉注射体外转染的成纤维细胞。与体外研究一样，体外转染的成纤维细胞在体内显示出高效的基因表达。用巨噬细胞特异性免疫染色分析组织切片。在成纤维细胞注射区域未见炎症迹象。本研究表明，FuGene6是一种高效的转染试剂，可用于原代人及兔成纤维细胞的体外非病毒转染以及体内治疗性非病毒基因递送。
