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Heparin can improve the viability of transfected cystic fibrosis cell lines in vitro

机译：肝素可改善体外转染的囊性纤维化细胞系的生存能力

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摘要

Cationic liposomes are widely used as gene transfer agents in in vitro and in vivo studies of cystic fibrosis. In this study we report comparative results of cationic mediated transfection in several cell lines.We have tested epithelial cell lines expressing the wild-type cystic fibrosis transmembrane protein CFTR (bronchial epithelium-16HBE14o-, submucosal gland-Calu3) and their cystic fibrosis counterparts (CFBE41o-, CFSMEo-), as well as baby hamster kidney fibroblast cell lines (BHK) heterologously expressing human CFTR. The cells were transfected with a green fluorescent protein plasmid complexed with commercial cationic liposome (Geneporter2, GP) and 25 kDa polyethylenimine (PEI). At the end of the incubation (2 hours), low molecular weight heparin was added in order to reduce the toxicity of the lipoplexes. Transfection efficiency and cell viability were measured by flow cytometry. Determination of fatty acid composition of cellular phospholipids was performed by capillary gas chromatography.The short incubation time was sufficient to obtain satisfactory transfection in all cell lines studied. Cells treated with PEI-complexes had lower transfection efficiency and viability compared to GP in all tested cell lines. DeltaF508 CFTR carrying airway epithelial cells were easier to transfect but had lower viability compared to their healthy counterparts. This was, however not the case for the BHK cells. The fatty acid analysis showed characteristic polyunsaturated fatty acid patterns, which correlated with the viability of the transfected cells.Low molecular mass heparin added at the end of the lipoplex incubation time could help to maintain the viability of the cells, without interfering with the transfection efficiency. (C) 2004 Elsevier Inc. All rights reserved.
机译：阳离子脂质体在囊性纤维化的体外和体内研究中广泛用作基因转移剂。在这项研究中，我们报告了阳离子介导的转染在几种细胞系中的比较结果。我们已经测试了表达野生型囊性纤维化跨膜蛋白CFTR（支气管上皮-16HBE14o-，粘膜下腺-Calu3）及其对应的囊性纤维化的上皮细胞系（ CFBE41o-，CFSMEo-），以及异源表达人类CFTR的仓鼠肾脏成纤维细胞系（BHK）。用绿色荧光蛋白质粒转染细胞，该质粒与市售阳离子脂质体（Geneporter2，GP）和25 kDa聚乙烯亚胺（PEI）复合。温育结束时（2小时），加入低分子量肝素以降低脂复合物的毒性。通过流式细胞术测量转染效率和细胞活力。通过毛细管气相色谱法测定细胞磷脂的脂肪酸组成。短的孵育时间足以在所有研究的细胞系中获得令人满意的转染。在所有测试的细胞系中，与GP相比，用PEI复合物处理的细胞的转染效率和活力较低。携带DeltaF508 CFTR的气道上皮细胞较其健康对应物更易于转染，但活力较低。但是，对于BHK细胞，情况并非如此。脂肪酸分析显示出特征性的多不饱和脂肪酸模式，与转染细胞的活力有关。在lipoplex孵育时间结束时添加低分子量肝素可以帮助维持细胞的活力，而不会影响转染效率。 （C）2004 Elsevier Inc.保留所有权利。

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