KERATINOCYTE GALVANOTAXIS AND WOUND HEALING

通过角质形成细胞的迁移使皮肤伤口再上皮化 作为伤口的重要和必要组成部分， 治愈这一提议提出了一个总体假设，即内生的 由受伤皮肤产生的电场提供了早期的线索， 定向角质细胞迁移。该提案建立在调查人员的 先前发现的阴极定向迁移（趋电性） 角质形成细胞中的电场相当于在哺乳动物中测量的电场 皮肤伤口。将要检验的假设是， 导致受体糖蛋白的横向电泳， 细胞膜的平面，引发不对称的信号事件 最终导致定向细胞迁移。不对称钙 内流被假设为该信号传导途径的一部分。 本研究的总体目标是确定人类 角质形成细胞趋电性具体目标将调查1）可能的 质膜靶向EGF受体，尿激酶PA受体，和 整合素; 2）调节Ca 2+内流; 3）下游信号激酶 4）角质形成细胞分化对趋电性的影响5） 电场参数最佳的趋电性。在这些研究中，3 将利用角质形成细胞迁移的模型，每个模型提供 独特的优势1)迁移单细胞的实时图像分析 提供了定量细胞速度、净易位和 细胞旅行的方向性。2)受伤的单层迁移提供了 关于角质形成细胞如何在受伤的邻接片的边缘移动的数据， 类似于正常表皮。3)一个受伤的“皮肤等效物”，或复合物 将成纤维细胞掺入胶原凝胶覆盖的培养系统 与分层培养的角质形成细胞，提供了机会， 在三维矩阵的背景下检查上皮再生， 表皮角质形成细胞和真皮细胞之间的细胞“串扰” 成纤维细胞 电刺激定向角质形成细胞迁移可能最终 提供了一种用于增强伤口愈合的强有力的治疗工具， 特别是在慢性、不愈合的伤口中。

Re-epithelialization of a skin wound by the migration of keratinocytes from the wound margin as an important and necessary component of wound healing. This proposal forwards the overall hypothesis that endogenous electric fields, generated by the wounded skin, provide an early cue for directed keratinocyte migration. The proposal builds on the investigators' previous finding of cathodally-directed migration (galvanotaxis) of keratinocytes in electric fields equivalent to those measured in mammalian skin wounds. The hypothesis that will be tested is that an electric field results in the lateral electrophoresis of receptor glycoproteins within the plane of the cell membrane, initiating asymmetrical signaling events which culminate in directed cellular migration. Asymmetrical calcium influx is hypothesized to be a part of this signaling pathway. The overall aim of the study is to define the mechanism of human keratinocyte galvanotaxis. Specific aims will investigate 1) possible plasma membrane targets the EGF receptor, the urokinase PA receptor, and integrins, 2) modulation of Ca 2+ influx 3) downstream signaling kinase pathways 4) effects of keratinocyte differentiation on galvanotaxis 5) the electric field parameters optimal for galvanotaxis. For these studies, 3 models of keratinocyte migration will be utilized, each offering a distinct advantage. 1) Real-time image analysis of migrating single cells provides the ability to quantitate cell velocity, net translocation and directedness of cell travel. 2) A wounded monolayer migration provides data on how keratinocytes move at the edge of wounded contiguous sheet, akin to normal epidermis. 3) A wounded "skin equivalent", or composite culture system with fibroblasts incorporated into collagen gel overlaid with a stratified cultured keratinocytes, provides an opportunity to examine re-epithelialization in the context of a 3-dimensional matrix and with cellular 'crosstalk' between epidermal keratinocyte and dermal fibroblast. Electrical stimulation of directed keratinocyte migration may ultimately provide a powerful therapeutic tool for enhancing wound healing, particularly in chronic, non-healing wounds.