MOLECULAR INTERACTIONS ON ASBESTOS AND MESOTHELIAL CELLS

石棉引起的疾病的一个主要部位是胸膜， 表现为炎症、纤维化或恶性肿瘤。 关键 了解石棉引起的胸膜疾病 青石棉纤维与间皮细胞的相互作用， 由于青石棉是与胸膜疾病最相关的纤维， 间皮细胞对石棉特别敏感。 近几 研究，青石棉的内化由间皮细胞已经 发现通过将纤维与 玻连蛋白是血清的主要粘附蛋白。我们建议的研究将 确定玻连蛋白涂层青石棉是否被认可 并通过玻连蛋白的特异性细胞表面受体内化， 兔胸膜间皮细胞以及这种相互作用是否参与 基因表达的激活信号。此外，我们的研究 将讨论石棉引起的活性氧是否有助于 纤维内化和/或基因激活。白细胞介素-8（IL-8），一种基因 以前发现的青石棉诱导中石棉 细胞，将作为基因激活的标记，和一种新的荧光 共聚焦显微镜技术将用于计数细胞内和 细胞外纤维分开。首先，青石棉和对照颗粒 用玻连蛋白包被，其它细胞外基质蛋白或血清将 与间皮细胞孵育以确定玻连蛋白是否 特别地增加青石棉的粘附和/或内化。 第二，两个间皮细胞表面玻连蛋白受体，α-v- β 3和α-v-β 5，将被特异性抗体阻断， 确定它们在介导间皮细胞附着到 玻连蛋白和介导间皮细胞内化 青石棉 其他玻连蛋白受体将寻求使用 免疫沉淀和亲和层析。 蜂窝 玻连蛋白受体的分布及其共定位 将使用免疫细胞化学鉴定青石棉。三是 将确定玻连蛋白受体在IL-8基因表达中的作用 通过测定兔IL-8蛋白和玻连蛋白受体后的信息， 通过与特异性抗体聚集的整合素被阻断或激活。 第四，将研究间皮衍生的活性氧物种， 在青石棉的内化过程中， 细胞和石棉诱导的IL-8基因表达的间皮细胞。 在这个建议中，青石棉纤维的特定相互作用 与间皮细胞的关系， 石棉毒性的机制

A major site of asbestos-induced disease is the pleura, where disease manifests itself as either inflammation, fibrosis or malignancy. Crucial to understanding asbestos-induced pleural disease is understanding the interaction of the crocidolite asbestos fiber with the mesothelial cell, since crocidolite is the fiber most associated with pleural disease and the mesothelial cell is particularly sensitive to asbestos. In recent studies, internalization of crocidolite by the mesothelial cell has been found to be increased significantly by incubating the fibers with vitronectin, the major adhesive protein of serum. Our proposed study will determine whether vitronectin-coated crocidolite asbestos is recognized and internalized via specific cell surface receptors for vitronectin on rabbit pleural mesothelial cells and whether this interaction participates in signaling the activation of gene expression. In addition, our study will address whether asbestos-induced active oxygen species contribute to fiber internalization and/or gene activation. Interleukin-8 (IL-8), a gene previously found to be induced by crocidolite asbestos in mesothelial cells, will serve as a marker of gene activation, and a novel fluorescence confocal microscopic technique will be used to count intracellular and extracellular fibers separately. First, crocidolite and control particles coated with vitronectin, other extracellular matrix proteins or serum will be incubated with mesothelial cells to determine whether vitronectin specifically increases adherence and/or internalization of crocidolite. Second, two mesothelial cell surface receptors for vitronectin, alpha-v- beta3 and alpha-v-beta5, will be blocked with specific antibodies to determine their role in mediating mesothelial cell attachment to vitronectin and in mediating mesothelial cell internalization of crocidolite. Additional vitronectin receptors will be sought using immunoprecipitation and affinity chromatography. The cellular distribution of vitronectin receptors and their possible colocalization with crocidolite will be identified using immunocytochemistry. Third, the role of vitronectin receptors in lL-8 gene expression will be determined by measuring rabbit lL-8 protein and message after vitronectin receptors are blocked or activated by integrin clustering with specific antibodies. Fourth, mesothelial-derived active oxygen species will be studied for an independent or contributory role in internalization of crocidolite by the cells and in asbestos-induced lL-8 gene expression by mesothelial cells. In this proposal, the specific interaction of crocidolite asbestos fibers with the mesothelial cell will be explored to provide new information about mechanisms of asbestos toxicity.