Innovative Bioengineering for Cutaneous Wounds

描述（由申请人提供）： 在这项转化研究计划中，我们测试了这样一个假设：通过移植由人类角质形成细胞产生的临时人类皮肤替代品，可以增强慢性皮肤伤口的愈合，这些替代品经过基因工程改造，可以增强下面伤口床的血管化。描述了实现这一目标的两种策略，I.) 血管生成生长因子 VEGF-165 的组织特异性表达和 II.) 组成型活性转录因子缺氧诱导因子-1α (HIF-1a) 的组织特异性表达。 HIF-1a 是细胞和全身氧稳态的通用调节剂，在伤口愈合过程中过度表达。已知 HIF 可以上调许多靶基因的表达，包括那些参与血管生成的基因。我们离体产生了基因工程人类角质形成细胞系，无论氧张力如何，它们都能以组织特异性方式持续产生 VEGF-165 和活性 HIF-1a 蛋白。使用组织工程策略，这些角质形成细胞系将用于生产具有生物活性的人类皮肤替代品，以研究持续表皮血管生成信号对血管化和伤口愈合的生理和/或病理后果。修订后的目标是： 具体目标 1. 分离稳定的 NIKS 角质形成细胞系（NIKS（空载体）、NIKS（VEGF165）、NIKS（HIF-1α/ODD）、NIKS（HIF-1α））。验证这些新 NIKS 细胞系的单层培养物产生和分泌增强水平的生物活性血管生成蛋白。具体目标 2. 使用基因工程 NIKS（空载体）、NIKS（VEGF-165）、NIKS（HIF-1α）和 NIKS（HIF-1α/ODD）角质形成细胞生产人类皮肤替代品，并分析靶基因表达和血管生成蛋白的产生。通过免疫组织化学分析基因工程组织的组织学特征。通过原位杂交和定量 PCR 确定 HIF-1a 靶基因表达。使用定量分析确认生物活性血管生成蛋白水平的提高。具体目标 3. 使用由 NIKS、NIKS（空载体）、NlKS（VEGF-165）、NlKS（HIF-1a）和 NIKS（HIF-1a/ODD）细胞系生成的人皮肤替代组织异种移植物确定组成性表皮血管生成信号对伤口愈合、生理和病理血管产生、组织和功能的影响。

DESCRIPTION (provided by applicant): In this translational research proposal we test the hypothesis that healing of chronic cutaneous wounds may be enhanced by grafting of temporary human skin substitutes generated from human keratinocytes which have been genetically engineered to enhance vascularization of the underlying wound bed. Two strategies to achieve this goal are described, I.) tissue-specific expression of the angiogenic growth factor VEGF-165 and II.) tissue-specific expression of a constitutively active transcription, factor hypoxia-inducible factor-1alpha (HIF-1a). HIF-1a is a universal regulator of cellular and systemic oxygen homeostasis and is overexpressed during wound healing. HIF is known to upregulate the expression of a number of target genes including those involved in vasculogenesis. We generated genetically engineered human keratinocyte cell lines ex vivo that constitutively produce VEGF-165 and active HIF-1a protein in a tissue-specific manner regardless of oxygen tension. Using tissue engineering strategies these keratinocyte cell lines will be used to produce biologically active human skin substitutes to investigate the physiological and/or pathological consequences of persistent epidermal angiogenic signaling on vascularization and wound healing. The revised aims are: Specific Aim 1. Isolate stable NIKS keratinocyte lines (NIKS(Empty Vector) , NIKS(VEGF165), NIKS(HIF-1alpha/ODD), NIKS(HIF-1alpha)).Verify that monolayer cultures of these new NIKS cell lines produce and secrete enhanced levels of biologically active angiogenic proteins. Specific Aim 2. Produce human skin substitutes using genetically engineered NIKS(Empty Vector), NIKS(VEGF-165), NIKS(HIF-1alpha), and NIKS(HIF-1alpha/ODD) keratinocytes and analyze for target gene expression and angiogenic protein production. Analyze the histological features of genetically engineered tissues by immunohistochemistry. Determine HIF-1a target gene expression by in situ hybridization and quantitative PCR. Confirm enhanced levels of biologically active angiogenic proteins using quantitative assays. Specific Aim 3. Determine the effects of constitutive, epidermal angiogenic signaling on wound healing, physiological and pathological vessel production, organization, and function using human skin substitute tissue xenografts generated from NIKS, NIKS(Empty Vector), NlKS(VEGF-165) , NlKS(HIF-1a), and NIKS(HIF-1a/ODD) cell lines.