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）： 在这项翻译研究建议中，我们检验了以下假设：通过嫁接暂时的人类皮肤替代品，可以通过基因工程来增强潜在伤口床的血管化来增强慢性皮肤伤口的愈合。描述了实现此目标的两种策略。 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表达。已知HIF可以上调许多靶基因，包括涉及血管生成的基因。我们在体内产生了基因工程的人角质形成细胞系，该细胞系具有组成性地生产VEGF-165和活性HIF-1A蛋白，无论氧气张力如何。使用组织工程策略，这些角质形成细胞系将用于产生具有生物活性的人皮替代物，以研究持续的表皮血管生成信号在血管化和伤口愈合方面的生理和/或病理后果。修订后的目的是：特定目标1。孤立稳定的角膜细胞线（NIKS（空载体），NIKS（VEGF165），NIKS（HIF-1ALPHA/ODD），NIKS（HIF-1ALPHA），验证这些新NIKS细胞系的单层培养和分泌性增强的量的单层培养物，并具有增强型的单层。具体目标2。使用基因工程的NIK（空载体），NIKS（VEGF-165），NIKS（HIF-1Alpha）和NIKS（HIF-1Alpha/Odd）角质形成细胞生产人皮肤替代品，并分析靶基因表达和血管生成蛋白。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。使用定量测定，确认生物活性血管生成蛋白的水平增强。具体目的3。确定组成型，表皮血管生成信号对使用人体皮肤替代组织的替代组织异种移植物产生的，NIKS（空载体），NLKS（VEGF-165），NLKS（nlks），NLKS（HIF-1A）（HIF-1A）和NIKS（HIF-1A）（HIF-1A）（niks）（hiff-165）（niks）（niks）（vegf-165）（niks）（vegf-165