Identification of an IL-6 induced keratinocyte motogen

描述(由申请人提供)： 在美国，每年有超过600万人患上慢性皮肤溃疡。长期以来，加强皮肤伤口愈合一直是卫生保健专业人员难以实现的目标。我们先前的研究表明，与野生型对照动物相比，白介素6缺陷转基因小鼠(IL-6KO)的皮肤伤口愈合明显延迟。虽然IL-6在牛皮癣等疾病中的作用得到了很好的证明，但对于这种细胞因子在伤口愈合等再生反应中可能发挥的作用却知之甚少。为了进一步研究IL-6在皮肤创面愈合中的作用，利用培养的新生IL-6KO小鼠表皮角质形成细胞和真皮成纤维细胞，建立了皮肤创面愈合的体外模型。该系统允许直接评估IL-6对皮肤细胞的影响，而不会混淆内源性IL-6的存在。使用这种培养系统，我们发现IL-6似乎显著地诱导培养的角质形成细胞的细胞运动。然而，这种效应似乎是间接的，需要与真皮成纤维细胞共同培养。初步的基因芯片实验没有表明诱导了一种已知的诱导角质形成细胞迁移的分泌性蛋白。 在这一应用中，我们建议：1)鉴定和鉴定IL-6诱导的真皮成纤维细胞产生的迁移因子。为此，将对暴露于IL-6的IL-6KO真皮成纤维细胞和暴露于IL-6/成纤维细胞条件培养液的表皮角质形成细胞进行进一步的基因芯片实验。IL-6/成纤维细胞条件培养液也将被浓缩，并进行离子交换层析。分离的IL-6KO角质形成细胞将暴露于从层析分离中收集的部分，并将评估迁移潜力。诱导迁移反应的组分将通过二维凝胶分离和胰酶指纹图谱进行蛋白质组学分析，并将使用MALDI-TOF质谱仪进行测定。一旦确定，将在从IL-6KO和野生型小鼠分离的角质形成细胞上评估迁移因子(S)的致癌潜力。这些实验的结果有望最终开发出一种有效的治疗慢性伤口的方法。

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.