DERMAL-EPIDERMAL INTERACTIONS IN DIABETIC WOUND HEALING

我们的长期目标是开发基于基因转移的治疗策略，以完全纠正糖尿病患者的伤口愈合损害。这项建议的总体目标是了解真皮-表皮相互作用正常情况下诱导伤口愈合中的再上皮化过程的机制，这一过程如何在糖尿病伤口愈合中失控，以及PDGF-B的过度表达如何“启动”真皮-表皮相互作用恢复伤口愈合过程。在我们实验室前期工作的基础上，我们提出了总体的工作假设：糖尿病创面具有紊乱的真皮-表皮相互作用，缺乏创伤愈合有序进行所需的正常水平和生长顺序，这种无序的真皮-表皮相互作用可被成纤维细胞产生的PDGF-B过度表达诱导的生长因子纠正，从而加速角质形成细胞的增殖和迁移。为了研究这一假说，我们设计了具有以下特定目的的实验：1.检验PDGF过表达诱导真皮至表皮信号的机制是通过在真皮成纤维细胞中诱导PDGF-A介导的假说；2.检验PDGF过表达诱导真皮至真皮信号的机制之一是通过在真皮成纤维细胞中诱导PDGF-A介导的假说；2.检验PDGF-B过表达纠正糖尿病受损角质形成细胞迁移和增殖的机制之一是诱导真皮成纤维细胞中转化生长因子-α的表达；III.验证PDGF-B过表达纠正糖尿病损伤的再上皮化中表皮相互作用的另一种机制是通过诱导真皮成纤维细胞产生纤维连接蛋白和角质形成细胞中α5bneta1整合素的表达。我们将使用糖尿病损伤创面愈合的体内模型，包括db/db小鼠模型、非肥胖糖尿病小鼠，以及转化生长因子-α基因敲除小鼠中链脲佐菌素诱导的糖尿病。此外，还将使用Transwell、共培养和器官型皮肤重建模型的体外技术。我们将通过标准病史、免疫细胞化学、原位杂交、免疫荧光染色和共聚焦显微镜、聚合酶链式反应、逆转录聚合酶链式反应、Western印迹和酶联免疫吸附试验分析生长因子过表达的影响。这些研究的结果将直接关系到将腺病毒介导的基因转移策略转化为治疗不可愈合的糖尿病足部溃疡，有可能改善这种疾病最常见的并发症之一。

Our long-term goal is to develop gene transfer based treatment strategies to completely correct the wound healing impairment in diabetes. The overall objective of this proposal is to understand the mechanisms by which dermal-epidermal interactions normally induce the process of re- epithelialization in wound healing, how this process is disregulated in diabetic wound healing, and how over-expression of PDGF-B can "jump start" dermal-epidermal interactions restoring the wound healing process. Based on preliminary work at our laboratory we have developed the overall working hypothesis that: diabetic wounds have disordered dermal-epidermal interactions lacking the normal level and sequence of growth necessary for the orderly progression of wound healing and this disordered dermal-epidermal interaction is corrected by PDGF-B over- expression-induced growth factors made by fibroblasts which accelerate keratinocyte proliferation and migration. To investigate this hypothesis we plan experiments with the following specific aims: I. To test the hypothesis that the mechanisms by which PDGF over-expression induces dermal signaling to the epidermis is mediated by induction of PDGF-A in dermal fibroblasts; II. To test the hypothesis that one mechanism by which PDGF over-expression induces dermal signaling to the epidermis is mediated by induction of PDGF-A in dermal fibroblasts; II. To test the hypothesis that one mechanism by which PDGF-B over-expression corrects diabetic impaired keratinocyte migration and proliferation is the induction of TGF-alpha expression in dermal fibroblasts; III. To test the hypothesis that another mechanism by which PDGF-B over-expression corrects-epidermal interactions in diabetes impaired re-epithelialization is by inducing production of fibronectin by dermal fibroblasts and alpha5bneta1 integrin expression in keratinocytes. We will use a combination in vivo models of diabetic impaired wound healing including the db/db mouse model, non-obese diabetic mice, and streptotozocin-inducing diabetes in TGF-alpha knock out mice. In addition, in vitro techniques using transwell, co-culture and organotypic skin reconstruct models will be used. We will analyze the effects of growth factor over-expression by standard history, immunocytochemistry, in situ hybridization, immunofluorescent staining and confocal microscopy, PCR, RT-PCR, Western blot, and ELISA. The results of these studies will have direct bearing on the translation of adenoviral mediated gene transfer strategies to the treatment of non- healing diabetic foot ulcers with the potential to ameliorate one of the most common complications of this disease.