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，以完全纠正糖尿病伤口愈合障碍。本提案的总体目标是了解真皮-表皮相互作用通常诱导伤口愈合中再上皮化过程的机制，该过程在糖尿病伤口愈合中如何失调，以及PDGF-B的过表达如何能够“跳跃启动”真皮-表皮相互作用恢复伤口愈合过程。基于我们实验室的初步工作，我们已经开发了总体工作假设：糖尿病伤口具有紊乱的真皮-表皮相互作用，缺乏伤口愈合有序进展所需的正常水平和生长顺序，并且这种紊乱的真皮-表皮相互作用通过由成纤维细胞产生的PDGF-B过表达诱导的生长因子来校正，所述生长因子加速角质形成细胞增殖和迁移。为了研究这一假设，我们计划了具有以下特定目标的实验：检验PDGF过度表达诱导真皮信号传导至表皮的机制是通过诱导真皮成纤维细胞中的PDGF-A介导的假设; II.为了检验以下假设，即PDGF过表达诱导表皮信号传导至表皮的一种机制是通过诱导真皮成纤维细胞中的PDGF-A介导的; II.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一种机制是诱导真皮成纤维细胞中TGF-α表达的假设; III.为了检验PDGF-B过表达纠正糖尿病损害的再上皮形成中的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连蛋白和角质形成细胞中的α 5 bneta 1整联蛋白表达的假设。我们将联合使用糖尿病损伤伤口愈合的体内模型，包括db/db小鼠模型、非肥胖糖尿病小鼠和TGF-α基因敲除小鼠中链脲霉素诱导的糖尿病。此外，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、PCR、RT-PCR、Western印迹和ELISA来分析生长因子过表达的影响。这些研究的结果将直接影响腺病毒介导的基因转移策略的翻译，以治疗非愈合性糖尿病足溃疡，并有可能改善这种疾病的最常见并发症之一。

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.