IMMUNE MEDIATED ALTERATIONS IN CONNECTIVE TISSUE

结缔组织中免疫介导的改变

• 批准号: 2517431
• 负责人: JOSEPH H KORN
• 金额: $ 27.52万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-10-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2517431
• 关键词: apoptosis; cell growth regulation; cellular immunity; clinical research; collagen; collagenase; connective tissue metabolism; cytotoxicity; fibroblasts; gel mobility shift assay; gene expression; human subject; molecular cloning; protein metabolism; scleroderma; tissue /cell culture; transcription factor; transfection

DESCRIPTION: (Adapted from the applicant's abstract) Systemic sclerosis (SSc) is characterized by the accumulation of excess collagen and other matrix proteins in the skin and other organs as well as widespread vascular pathology and immune cell activation. The investigators' studies have focused on understanding the basis for excessive matrix biosynthesis. They have used, as a working hypothesis, the concept that immune or other events, in addition to stimulating fibroblasts, lead to clonal selection of high collagen producing fibroblast subpopulations which then manifest a fibrotic phenotype. The investigators have shown that fibroblasts are heterogeneous in their proliferative capacity, response to cytokines, and synthesis of collagen, matrix metalloproteinases, and other products. This heterogeneity was present at both the protein and mRNA level and that it represented a stable clonal phenotype. The investigators showed that immune cell products, in vitro, can lead to long-term alterations in fibroblast phenotype and that the clonal makeup of fibroblasts is skewed towards high collagen producing cells in SSc. Finally, they cloned the human cDNA and genomic DNA for a collagen transcription factor, cKROX. The investigators wish to continue and extend these studies to gain further understanding of the pathophysiology of fibrotic disease. In the next project period, they propose to explore further mechanisms leading to fibroblast abnormalities in SSc. They will investigate the human collagen transcription factor, cKROX, that they have cloned, and examine its expression in SSc and in clonal populations, as well as its regulation. They will examine differences in gene expression between scleroderma-involved and -uninvolved skin fibroblasts using differential display. They hope to identify among the expressed genes detected by differential display possible regulatory genes underlying abnormal fibroblast metabolism. They will test the hypothesis that apoptosis plays a role in leading to selective overgrowth of fibroblast populations in SSc. They will determine whether SSc fibroblasts have abnormal responses to apoptotic signals and whether these are based on different expression of proteins important in apoptosis such as Fas, bcl-2, p53, and c-myc. They will look at the role that hypoxia might play in altering fibroblast metabolism in SSc. They anticipate that these studies will improve the understanding of the interactions of fibroblasts, their milieu in inflammatory states, in general, and in SSc, in particular. The first specific aim is to examine the possibility that differences in the synthesis of collagen between SSc and normal cells are due to differential expression of cKROX. Stable transfectants will be used to evaluate cKROX as a transcription factor. Known factors that regulate collagen synthesis will be evaluated for their influence on cKROX expression. Lesional skin, nonlesional skin, normal skin, and clonal populations of fibroblasts will be evaluated using gel shift analysis, Northern blots, and RT-PCR for cKROX expression. In the second specific aim, differential display libraries will be developed from cloned and normal fibroblasts and lesional and nonlesional fibroblasts. The genes identified will be cloned and characterized. The third specific aim is to evaluate selective influences for clonal proliferation of fibroblasts including apoptosis and hypoxia.

描述：（改编自申请人摘要）系统性硬化症 (SSc)其特征在于过量胶原蛋白和其他 皮肤和其他器官中的基质蛋白以及广泛的血管 病理学和免疫细胞活化。 研究人员的研究 专注于理解过度基质生物合成的基础。 他们 作为一个工作假设，使用了免疫或其他事件的概念， 除了刺激成纤维细胞外，还导致较高细胞克隆选择 产生胶原的成纤维细胞亚群， 表型 研究人员已经表明，成纤维细胞是异质性的， 在它们的增殖能力、对细胞因子的反应和 胶原蛋白、基质金属蛋白酶和其他产品。 这种异质性 在蛋白质和mRNA水平上都存在，它代表了一种 稳定克隆表型。 研究人员发现， 产品，在体外，可导致成纤维细胞的长期改变， 表型和成纤维细胞的克隆组成偏向高 SSc中的胶原生成细胞。 最后，他们克隆了人类cDNA， 胶原转录因子cKROX的基因组DNA。 调查人员 我希望继续和扩大这些研究，以进一步了解 纤维变性疾病的病理生理学。 在下一个项目期间，他们建议探讨进一步的机制 导致SSc中的成纤维细胞异常。 他们会调查人类 胶原蛋白转录因子cKROX，他们已经克隆，并检查其 在SSc和克隆群体中的表达，以及其调节。 他们将研究在基因表达上的差异， 硬皮病累及和未累及皮肤成纤维细胞的分化 显示. 他们希望通过检测到的表达基因中， 差异显示可能的调节基因异常 成纤维细胞代谢 他们将检验细胞凋亡在细胞凋亡中起重要作用的假设。 在导致SSc中成纤维细胞群体选择性过度生长中的作用。 他们将确定SSc成纤维细胞是否对 凋亡信号，以及这些信号是否基于 在凋亡中重要的蛋白，如Fas、bcl-2、p53和c-myc。 他们 将研究缺氧可能在改变成纤维细胞中发挥的作用， SSc中的代谢。 他们预计，这些研究将改善 了解成纤维细胞的相互作用，它们在 炎症状态，特别是在SSc中。 第一个具体目标是审查的可能性，差异， SSc和正常细胞之间胶原蛋白合成是由于差异 cKROX的表达。 稳定的转染子将用于评估cKROX， 转录因子。 调节胶原蛋白合成的已知因素将 评价其对cKROX表达的影响。 皮肤损伤， 非病变皮肤、正常皮肤和成纤维细胞的克隆群体将被 使用凝胶位移分析、北方印迹和RT-PCR评价cKROX 表情 在第二个具体目标中，将开发差异显示文库 来自克隆的和正常的成纤维细胞以及损伤的和非损伤的成纤维细胞。 将对鉴定的基因进行克隆和鉴定。 第三个具体目标是评估克隆的选择性影响， 成纤维细胞增殖，包括凋亡和缺氧。