Department of Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
Wound Repair Regen. 2012 May-Jun;20(3):414-24. doi: 10.1111/j.1524-475X.2012.00786.x.
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 beta 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 (NIKS(hBD) (-3) ) using nonviral methods. Levels of hBD-3 mRNA and protein in three-dimensional skin tissue produced from NIKS(hBD) (-3) were determined using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Tissue architecture was characterized by hematoxylin and eosin staining and by indirect immunofluorescence 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 NIKS(hBD) (-3) or control NIKS possessed histologic features of interfollicular epidermis and exhibited normal tissue growth and differentiation. NIKS(hBD) (-3) tissue contained approximately fivefold more hBD-3 protein than tissue containing unmodified control NIKS. In vitro studies showed that NIKS(hBD) (-3) tissue produced a significant reduction in the growth of Staphylococcus aureus multiple peptide resistance factor (mprF) compared with control tissue. In an in vivo infected murine burn model, NIKS(hBD) (-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.
先天免疫系统可调节宿主防御肽的表达，以在伤口愈合过程中抵御感染。我们通过增强角质形成细胞中宿主防御肽人β防御素-3（hBD-3）的表达，生成一种三维生物敷料，以改善感染性伤口的愈合。使用非病毒方法，通过含有表皮特异性启动子驱动 hBD-3 的构建体（NIKS(hBD)-3），将 NIKS 人角质形成细胞系进行体外稳定转染。通过定量聚合酶链反应和酶联免疫吸附试验分别测定 NIKS(hBD)-3 产生的三维皮肤组织中的 hBD-3 mRNA 和蛋白水平。通过苏木精和伊红染色以及增殖和角质形成细胞分化标志物的间接免疫荧光来表征组织结构。通过体外细菌生长测定和体内使用小鼠烧伤感染模型来评估抗菌活性。含有表皮 NIKS(hBD)-3 或对照 NIKS 的三维全厚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。NIKS(hBD)-3 组织中的 hBD-3 蛋白含量比含有未修饰对照 NIKS 的组织高约五倍。体外研究表明，NIKS(hBD)-3 组织产生的金黄色葡萄球菌多肽耐药因子（mprF）生长减少与对照组织相比具有统计学意义。在体内感染的小鼠烧伤模型中，NIKS(hBD)-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 可导致动物感染三度烧伤模型中金黄色葡萄球菌菌株的生长受到治疗相关的减少。
