CULTURED EPIDERMAL GRAFTS FOR BURN PATIENTS

描述（由申请人提供）：在过去的资助期内， 实验室已经研究了表皮生物学的基础和临床方面。 我们的临床研究集中在影响伤口修复的内在因素。 基础研究集中在受控蛋白水解的重要性， 特别是纤溶酶级联，对细胞增殖、迁移和基质 与伤口修复和纤维化相关的重塑。我们已经澄清了 纤溶酶原激活物抑制剂I型（PAl-I），主要的 纤溶酶生成的负调节因子，在以下方面发挥调节作用： 控制a）细胞迁移，B）伤口修复，c）纤维化， 和d）对生长因子和血清的反应。我们已经证明， PAI-1表达的扰动显著损害了细胞凋亡的程度和速率。 伤口闭合初步研究还表明，硝酸镓（GN）， III组过渡元素，诱导真皮成纤维细胞上调 整合素表达和细胞外基质合成。此外，GN 加速体内伤口修复。考虑到PAI-1（和PAI-2）的重要性， 纤溶酶和金属蛋白酶级联的其他成员）， 伤口床内和细胞运动中平衡的蛋白水解环境， 在分子水平上理解PAl-I基因控制可能会导致重要的 旨在改善不愈合状态的损伤修复新见解 慢性创伤 我们建议实现以下目标： I.确定PAl-I或uPA合成的遗传诱导的调节 平衡影响真皮成纤维细胞粘附，迁移， 简单和复杂基质的降解。 二.定义诱导整合素亚基的分子机制 在GN处理的表达：A.表皮角质形成细胞，以评估GN如何诱导 基底角质形成细胞呈现活化和能动的表型， 表皮创伤修复的体外模型，和B.真皮成纤维细胞，以评估 如果GN诱导活化表型，并确定ON是否调节 在基质依赖性运动的体外模型中的成纤维细胞运动，和 无论这是依赖于蛋白酶或抑制剂的诱导。 三.利用肥胖db/db糖尿病小鼠模型评估 蛋白水解平衡对伤口修复的影响：A.评估外源性 施用重组PAl-I、TIMP-1和TIMP-2蛋白以及化学制剂 金属蛋白酶、硝酸镓和血管生成介质的抑制剂 (e.g., VEGF、FGF、Ang 1和Ang 2）对创面修复时间进程的影响; B. 确定用uPA、PAl-I、TIMP-1、TIMP-1、TIMP-2转染伤口区域的效果。 TIMP-2和血管生成介质对创伤修复的影响

DESCRIPTION (provided by applicant): During the past granting period, our laboratories have studied the basic and clinical aspects of epidermal biology. Our clinical studies focused on intrinsic factors that affect wound repair. Basic investigations focused on the importance of controlled proteolysis, in particular the plasmin cascade, on cell proliferation, migration and matrix remodeling associated with wound repair and fibrosis. We have clarified the mechanisms by which Plasminogen Activator Inhibitor Type I (PAl-I), the major negative regulator of plasmin generation, exerts a regulatory role in controlling the processes of a) cell migration, b) wound repair, c) fibrosis, and d) response to growth factors and serum. We have shown that molecular perturbation of PAI-l expression markedly impairs both the extent and rate of wound closure. Preliminary studies have also shown that gallium nitrate (GN), a group III transitional element, induces dermal fibroblasts to upregulate integrin expression and extracellular matrix synthesis. Additionally, GN accelerates wound repair in vivo. Given the emerging importance of PAI-l (and other members of the plasmin and metalloproteinase cascades) in maintenance of a balanced proteolytic environment within the wound bed and in cell locomotion, understanding PAl-I gene control at the molecular level may lead to important new insights into injury repair aimed at improving the state of non-healing chronic wounds. We propose to address the following aims: I. Determine whether genetically induced modulation of PAl-I or uPA synthetic balance affects dermal fibroblast adhesion to, migration through and degradation of simple and complex matrices. II. Define the molecular mechanism underlying induced integrin subunit expression in GN- treated: A. epidermal keratinocytes, to assess how GN induces the basal keratinocytes to assume an activated and motile phenotype using our in vitro model of epidermal wound repair, and B. dermal fibroblasts, to assess if GN induces an activated phenotype, and to determine if ON modulates fibroblast motility in an in vitro model of matrix dependent motility, and whether this is dependent on induction of proteases or inhibitors. III. Utilize the obese db/db diabetic mouse model to assess the influence of proteolytic balance on wound repair by: A. Assessing the effect of exogenously administered recombinant PAl-I, TIMP-1 and TIMP-2 proteins as well as chemical inhibitors of metalloproteinases, gallium nitrate and angiogenic mediators (e.g., VEGF, FGF, Ang1 and Ang2) on the time course of wound repair, and B. Determining the effects of wound field transfection with uPA, PAl-I, TIMP-1, TIMP-2 and angiogenic mediators on wound repair.