Identification of an IL-6 induced keratinocyte motogen

在美国，每年有超过600万人患慢性皮肤溃疡。的加持 皮肤伤口愈合长期以来一直是卫生保健专业人员难以实现的目标。我们以前的研究 表明白细胞介素-6缺陷型转基因小鼠（IL-6KO）表现出显著延迟皮肤创伤 与野生型对照动物相比的愈合。虽然IL-6在疾病中的作用已有很好的记录， 在银屑病等疾病中，这种细胞因子可能在再生障碍中发挥的作用知之甚少。 如伤口愈合。为了进一步描述IL-6在皮肤伤口愈合中的作用，在体外， 模型是利用培养的表皮角质形成细胞和真皮成纤维细胞从新生儿IL- 6KO小鼠。该系统允许直接评估IL-6对皮肤细胞的作用，而不需要使用免疫组织化学方法。 混杂内源性IL-6的存在。使用这种培养系统，我们发现IL-6似乎 在培养角质形成细胞中显著诱导细胞运动。然而，这种影响似乎是间接的， 需要与真皮成纤维细胞共培养。初步的基因阵列实验并没有表明 已知诱导角质形成细胞迁移的分泌蛋白的诱导。 在本申请中，我们提出：1）表征和2）鉴定IL-6诱导的真皮成纤维细胞产生的 迁移因素为了做到这一点，将用IL-6KO真皮进行进一步的基因阵列实验。 暴露于IL-6的成纤维细胞和暴露于IL-6/成纤维细胞条件培养基的表皮角质形成细胞。白介素- 6/成纤维细胞条件培养基也将被浓缩，并且离子交换色谱将被纯化。 执行。将分离的IL-6KO角质形成细胞暴露于从层析柱收集的级分。 将评估分离和迁移潜力。诱导迁移反应的组分将 采用2D凝胶分离和胰蛋白酶指纹图谱进行蛋白质组学分析， MALDI-TOF质谱仪。一旦确定，迁移因子的运动发生潜力将是 对来自IL-6KO和野生型小鼠的分离的角质形成细胞进行评估。这些实验的结果将 有望最终开发出一种治疗慢性伤口的有效方法。

In the United States, over 6 million individuals develop chronic skin ulcers annually. The augmentation of cutaneous wound healing has long been an elusive goal for health care professionals. Our previous studies indicate that interleukin-6 deficient transgenic mice (IL-6KO) display significantly delayed cutaneous wound healing compared to wild type control animals. While the role of IL-6 is well documented in disease conditions such as psoriasis, little is known about the role this cytokine might play in regenerative responses such as wound healing. To further describe the role of IL-6 in skin wound healing, an in vitro model was developed utilizing cultured epidermal keratinocyte and dermal fibroblast cells from neonatal IL- 6KO mice. This system allows for the direct assessment of the effects of IL-6 on skin cells without the confounding presence of endogenous IL-6. Using this culture system we have found that IL-6 appears to significantly induce cell motility, in cultured keratinocytes. However, this effect appears to be indirect and requires co-culture with dermal fibroblasts. Preliminary gene array experiments do not indicate the induction of a secreted protein known to induce keratinocyte migration. In this application we propose to: 1) characterize and 2) identify the IL-6-induced dermal fibroblast produced migratory factor. To do this, further gene array experiments will be conducted with IL-6KO dermal fibroblasts exposed to IL-6, and epidermal keratinocytes exposed to IL-6/fibroblast conditioned media. IL- 6/fibroblast conditioned media will also be concentrated, and ion exchange chromatography will be performed. Isolated IL-6KO keratinocytes will be exposed to fractions collected from the chromatographic separations, and migratory potential will be assessed. Fractions that induce a migratory response will be subject to proteomic analysis utilizing 2D gel seperation and tryptic fingerprinting will be determined with a MALDI-TOF mass spectrometer. Once identified, the motogenic potential of migratory factor(s) will be assessed on isolated keratinocytes from IL-6KO and wild type mice. The results of these experiments will hopefully lead to the eventual development of a useful treatment for chronic wounds.