MOLECULAR INTERACTIONS ON ASBESTOS AND MESOTHELIAL CELLS

石棉引起的疾病的一个主要部位是胸膜，在那里疾病 表现为炎症、纤维化或恶性。至关重要 了解石棉引起的胸膜疾病就是了解 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联系最密切的纤维， 间皮细胞对石棉特别敏感。在最近 研究表明，间皮细胞对青石棉的内化作用 发现通过与纤维孵化显著增加 Vitronectin，血清的主要黏附蛋白。我们建议的研究将 确定涂有玻璃胶的青石棉是否被识别 并通过特定的细胞表面玻璃连结蛋白受体内化 兔胸膜间皮细胞及其相互作用是否参与 在发出基因表达激活的信号方面。另外，我们的研究 将讨论石棉诱导的活性氧物种是否有助于 纤维内化和/或基因激活。白介素8，一种基因 先前发现间皮细胞中由青石棉诱导 细胞，将作为基因激活的标志，以及一种新的荧光 将使用共聚焦显微镜技术来计数细胞内和 胞外纤维分开。第一，青石棉和对照颗粒 包被玻璃连蛋白，其他细胞外基质蛋白或血清将 与间皮细胞孵育以确定玻璃连结蛋白 特别增加青石棉的粘附性和/或内在化。 第二，两种间皮细胞表面的玻璃连结蛋白受体，α-v-。 Beta3和α-v-Beta5，将被特异性抗体阻断 确定它们在介导间皮细胞粘附中的作用 Vitronectin及其在介导间皮细胞内化中的作用 青石棉。将寻找更多的Vitronectin受体 免疫沉淀和亲和层析。蜂窝手机 玻璃连结蛋白受体的分布及其可能的共定位 将通过免疫细胞化学鉴定青石棉。第三， 玻璃连结蛋白受体在IL-8基因表达中的作用将被确定 通过检测兔IL-8蛋白和玻璃体连接蛋白受体后的信息 通过与特定抗体的整合素聚集而被阻断或激活。 第四，将对间皮细胞衍生的活性氧物种进行研究 在青石棉内化中的独立或贡献作用 石棉诱导间皮细胞表达IL-8基因。 在这项建议中，青石棉纤维的特定相互作用 将为间皮细胞的探索提供新的信息 关于石棉毒性的机制。

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.