Identification of an IL-6 induced keratinocyte motogen

描述（由申请人提供）： 在美国，每年有超过600万人患慢性皮肤溃疡。皮肤伤口愈合的增强长期以来一直是医疗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-6 KO）显示显着延迟皮肤伤口愈合相比，野生型对照动物。虽然IL-6的作用在银屑病等疾病中有很好的记录，但对这种细胞因子在伤口愈合等再生反应中可能发挥的作用知之甚少。为了进一步描述IL-6在皮肤伤口愈合中的作用，利用来自新生IL-6 KO小鼠的培养的表皮角质形成细胞和真皮成纤维细胞开发了体外模型。该系统允许直接评估IL-6对皮肤细胞的作用，而不存在内源性IL-6的混淆。使用该培养系统，我们发现IL-6似乎在培养的角质形成细胞中显著诱导细胞运动。然而，这种作用似乎是间接的，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子。为此，将用暴露于IL-6的IL-6 KO真皮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进行进一步的基因阵列实验。还将对IL- 6/成纤维细胞条件培养基进行浓缩，并进行离子交换层析。将分离的IL-6 KO角质形成细胞暴露于从色谱分离中收集的级分，并评估迁移潜力。诱导迁移反应的组分将使用2D凝胶分离进行蛋白质组学分析，并使用MALDI-TOF质谱仪测定胰蛋白酶指纹图谱。一旦鉴定，将在来自IL-6 KO和野生型小鼠的分离的角质形成细胞上评估迁移因子的运动发生潜力。这些实验的结果将有望最终开发出一种有效的慢性伤口治疗方法。

DESCRIPTION (provided by applicant): 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.