Innovative Bioengineering for Cutaneous Wounds

针对皮肤伤口的创新生物工程

• 批准号: 6928052
• 负责人: B LYNN ALLEN-HOFFMANN
• 金额: $ 35.61万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6928052
• 关键词: angiogenesis; angiogenesis factor; athymic mouse; bioengineering /biomedical engineering; biological signal transduction; cell line; clinical research; gene expression; genetic manipulation; human tissue; hypoxia inducible factor 1; immunocytochemistry; in situ hybridization; keratinocyte; polymerase chain reaction; tissue /cell culture; tissue engineering; transcription factor; vascular endothelial growth factors; wound healing

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.

描述（由申请人提供）： 在这项转化研究提案中，我们测试了一种假设，即慢性皮肤伤口的愈合可能会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来增强，这些皮肤替代品已被基因工程改造以增强底层伤口床的血管化。两个战略，以实现这一目标的描述，一）。血管生成生长因子VEGF-165的组织特异性表达和II.）缺氧诱导因子-1 α（HIF-1a）的组成型活性转录的组织特异性表达。HIF-1a是细胞和全身氧稳态的通用调节剂，并且在伤口愈合期间过表达。已知HIF上调许多靶基因的表达，包括参与血管发生的那些。我们产生了基因工程改造的人角质形成细胞系离体组成型产生VEGF-165和活性HIF-1a蛋白在组织特异性的方式，无论氧张力。使用组织工程策略，这些角质形成细胞系将用于产生生物活性的人类皮肤替代品，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。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（VEGF 165）、NIKS（HIF-1 α/ODD）、NIKS（HIF-1 α））。验证这些新NIKS细胞系的单层培养物产生并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-1 alpha）和NIKS（HIF-1 alpha/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。免疫组化分析基因工程组织的组织学特征。原位杂交和定量PCR检测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的增强水平。具体目标3。使用由NIKS、NIKS（空载体）、NlKS（VEGF-165）、NlKS（HIF-1 a）和NIKS（HIF-1 a/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传导对伤口愈合、生理和病理血管生成、组织和功能的影响。