Innovative Bioengineering for Cutaneous Wounds

描述(由申请人提供)： 在这个翻译研究方案中，我们测试了这样一个假设，即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，可以促进慢性皮肤伤口的愈合，这些人角质形成细胞已经被基因工程改造，以增强伤口床下的血管形成。描述了实现这一目标的两种战略，I)血管生成生长因子VEGF-165和II的组织特异性表达。)组织特异性转录，低氧诱导因子-1α(HIF-1a)。HIF-1a是细胞和全身氧平衡的通用调节器，在伤口愈合过程中过度表达。众所周知，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。我们在体外产生了基因工程的人类角质形成细胞系，这些细胞系以组织特异性的方式结构性地产生血管内皮生长因子-165和活性的HIF-1a蛋白，而与氧分压无关。利用组织工程技术，这些角质形成细胞系将被用来制造具有生物活性的人类皮肤替代物，以研究持续的表皮血管生成信号对血管形成和伤口愈合的生理和/或病理后果。具体目的：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))，证实这些新的NIKS细胞株的单层培养能够产生和分泌高水平的生物活性血管生成蛋白。具体目的2.利用基因工程的NIKS(空载体)、NIKS(血管内皮生长因子-165)、NIKS(HIF-1α)和NIKS(HIF-1α/ODD)角质形成细胞制备人皮肤替代物，并分析靶基因表达和血管生成蛋白的产生。用免疫组织化学方法分析基因工程组织的组织学特征。用原位杂交和定量聚合酶链式反应检测HIF-1a靶基因的表达。使用定量分析确认生物活性血管生成蛋白水平的提高。具体目的3.利用从NIKS、NIKS(空载体)、N1KS(血管内皮生长因子-165)、N1KS(HIF-1a)和NIKS(HIF-1a/ODD)细胞系产生的人皮肤替代组织异种移植，确定结构性、表皮血管生成信号对伤口愈合、生理和病理血管生成、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.