Nonviral human beta defensin-3 expression in a bioengineered human skin tissue: a therapeutic alternative for infected wounds.
Nonviral human beta defensin-3 expression in a bioengineered human skin tissue: a therapeutic alternative for infected wounds.
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DOI：
10.1111/j.1524-475x.2012.00786.x
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发表时间：
2012-05
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影响因子：
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通讯作者：
Allen-Hoffmann BL
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作者：
Gibson AL;Thomas-Virnig CL;Centanni JM;Schlosser SJ;Johnston CE;Van Winkle KF;Szilagyi A;He LK;Shankar R;Allen-Hoffmann BL
先天免疫系统差异性地调节宿主防御肽的表达，以对抗伤口愈合过程中的感染。我们增强了角质形成细胞中宿主防御肽、人β防御素-3 (hBD-3)的表达，以生成三维生物敷料，以改善感染伤口的愈合。使用非病毒方法，用含有驱动 hBD-3 的表皮特异性启动子 (NIKShBD-3) 的构建体离体稳定转染 NIKS 人角质形成细胞系。分别使用定量 PCR 和 ELISA 测定由 NIKShBD-3 产生的三维皮肤组织中 hBD-3 mRNA 和蛋白质的水平。通过苏木精和伊红染色以及使用增殖和角质形成细胞分化标记物的间接免疫荧光来表征组织结构。使用体外细菌生长测定和体内小鼠烧伤感染模型评估抗菌活性。含有表皮NIKShBD-3或对照NIKS的三维全层皮肤组织具有滤泡间表皮的组织学特征，并表现出正常的组织生长和分化。 NIKShBD-3 组织含有的 hBD-3 蛋白比含有未修饰对照 NIKS 的组织多大约 5 倍。体外研究表明，与对照组织相比，NIKShBD-3 组织显着减少了金黄色葡萄球菌 (S. aureus) mprF 的生长。在体内感染的小鼠烧伤模型中，NIKShBD-3 组织导致细菌生长减少 90%。这些结果表明，通过生物工程皮肤组织持续递送 hBD-3 可导致感染三度烧伤伤口动物模型中金黄色葡萄球菌菌株生长的治疗相关减少。
The innate immune system differentially regulates the expression of host defense peptides to combat infection during wound healing. We enhanced the expression of a host defense peptide, human β defensin-3 (hBD-3), in keratinocytes to generate a three-dimensional biologic dressing to improve healing of infected wounds. The NIKS human keratinocyte cell line was stably transfected ex vivo with a construct containing an epidermis-specific promoter driving hBD-3(NIKShBD-3) using non-viral methods. Levels of hBD-3 mRNA and protein in three-dimensional skin tissue produced from NIKShBD-3 were determined using quantitative PCR and ELISA, respectively. Tissue architecture was characterized by hematoxylin and eosin staining, and by indirect immunofluorence using proliferation and keratinocyte differentiation markers. Antimicrobial activity was assessed using an in vitro bacterial growth assay and in vivo using a murine burn infection model. Three-dimensional full thickness skin tissues containing epidermal NIKShBD-3 or control NIKS possessed histologic features of interfollicular epidermis and exhibited normal tissue growth and differentiation. NIKShBD-3 tissue contained approximately 5-fold more hBD-3 protein than tissue containing unmodified control NIKS. In vitro studies showed that NIKShBD-3 tissue produced a significant reduction in the growth of Staphylococcusaureus (S. aureus) mprF compared to control tissue. In an in vivo infected murine burn model, NIKShBD-3 tissue resulted in a 90% reduction in bacterial growth. These results demonstrate that sustained delivery of hBD-3 by a bioengineered skin tissue results in a therapeutically relevant reduction in growth of a S. aureus strain in an animal model of infected third degree burn wounds.