Nonviral transfer of genes to pig primary keratinocytes. Induction of angiogenesis by composite grafts of modified keratinocytes overexpressing VEGF driven by a keratin promoter
Nonviral transfer of genes to pig primary keratinocytes. Induction of angiogenesis by composite grafts of modified keratinocytes overexpressing VEGF driven by a keratin promoter
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将基因非病毒转移至猪原代角质形成细胞。
DOI：
10.1038/sj.gt.3300986
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发表时间：
1999
期刊：
影响因子：
5.1
通讯作者：
J. Jorcano
中科院分区：
文献类型：
--
作者：
M. Río;F. Larcher;Á. Meana;J. Segovia;Antonio Barba Alvarez;J. Jorcano
培养的皮肤上皮移植已被证明在治疗大面积皮肤损失方面是救命的。很明显，这项技术的主要困难之一是由于真皮床严重损坏，移植物的整体接受率很低。皮肤替代物提供了培养的角质形成细胞作为表皮层，以及真皮类似物，为皮肤修复提供了更合适的材料。体外将促进基质再生的基因转移到角质形成细胞似乎是改善这些移植物的治疗作用的一个有吸引力的策略。在转基因小鼠的研究中，使用表皮特异性启动子作为外源基因的表达驱动因素，导致了高表达水平和层特异性。目前大多数原代角质形成细胞的基因转移方案包括用逆转录病毒载体转导表皮细胞。然而，含有这些长DNA片段启动子的基因构建体不能通过病毒转导实现。在这篇文章中，我们描述了一种包括脂质介导的转染法、G418选择和基于增强型绿色荧光蛋白(EGFP)的富集法，以获得高水平表达转基因的原代角质形成细胞。利用该方法，将5.2kb牛角蛋白K5启动子驱动的血管内皮细胞有丝分裂原血管内皮生长因子(VEGF)的基因稳定地导入猪的原代角质形成细胞。转基因角质形成细胞在含有纤维蛋白的活成纤维细胞凝胶上扩增，作为复合材料移植到裸鼠体内，新鲜组织检查和CD31免疫染色表明，转基因角质形成细胞在宿主基质中引发了强烈的血管生成反应。由于形成血管良好的创面床是伤口永久闭合的关键步骤，因此使用“血管生成”复合材料可能会改善创面床的准备和培养的角质形成细胞移植物的覆盖率。
Cultured epithelial grafts have proven to be life-saving in the treatment of large skin losses. It has become apparent that one of the main difficulties of this technology is the overall poor take of the grafts as a consequence of severely damaged dermal beds. Skin substitutes providing both cultured keratinocytes, as an epidermal layer, and a dermal analogous offer a more suitable material for skin repair. Ex vivo transfer of stroma regeneration-promoting genes to keratinocytes appears to be an attractive strategy for improving the therapeutic action of these grafts. The use of epidermal-specific promoters as expression drivers of exogenous genes results in both high expression levels and stratum specificity, as shown in transgenic mice studies. Most current gene transfer protocols to primary keratinocytes involve transduction of epidermal cells with retroviral vectors. However, transfer of gene constructs harboring these long DNA fragment promoters cannot be achieved through viral transduction. In this paper, we describe a protocol consisting of lipid-mediated transfection, G418 selection and an enhanced green fluorescence protein (EGFP)-based enrichment step for obtaining high levels of transgene-expressing primary keratinocytes. Using this protocol, the cDNA for vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen driven by the 5.2 kb bovine keratin K5 promoter, was stably transfected into pig primary keratinocytes. Genetically modified keratinocytes, expanded on live fibroblast-containing fibrin gels and transplanted to nude mice as a composite material, elicited a strong angiogenic response in the host stroma as determined by fresh tissue examination and CD31 immunostaining. Since the formation of a well-vascularized wound bed is a crucial step for permanent wound closure, the use of an ‘angiogenic’composite material may improve wound bed preparation and coverage with cultured keratinocyte grafts.