IMMUNE MEDIATED ALTERATIONS IN CONNECTIVE TISSUE

描述：(改编自申请者的摘要)系统性硬化症 (SSC)的特点是过量的胶原蛋白和其他 皮肤和其他器官以及广泛分布的血管中的基质蛋白 病理和免疫细胞激活。调查人员的研究表明 重点了解过度基质生物合成的基础。他们 使用免疫或其他事件的概念作为工作假说， 除了刺激成纤维细胞外，还能导致克隆性选择高 产生胶原的成纤维细胞亚群，然后表现为纤维化 表型。研究人员已经证明成纤维细胞是异质的。 在它们的增殖能力、对细胞因子的反应和合成 胶原蛋白、基质金属蛋白酶等产品。这种异质性 在蛋白质和信使核糖核酸水平上都存在，并且它代表了 稳定的克隆表型。研究人员发现，免疫细胞 在体外，产品可能会导致成纤维细胞的长期变化 表型和成纤维细胞的克隆性构成向高水平倾斜 SSC中的胶原生成细胞。最后，他们克隆了人类的cdna，并 胶原转录因子cKROX的基因组DNA。调查人员 希望继续和扩大这些研究，以进一步了解 纤维化疾病的病理生理学。 在下一个项目期，他们提议探索进一步的机制 导致SSC成纤维细胞异常。他们将调查人类 他们克隆的胶原转录因子cKROX，并检测其 在SSC和克隆种群中的表达及其调控。 他们将研究基因表达的差异 硬皮病受累皮肤成纤维细胞与非硬皮病皮肤成纤维细胞的差异 展示。他们希望在检测到的表达基因中识别出 差异显示异常可能的调控基因 成纤维细胞代谢。他们将检验这样一种假设，即细胞凋亡在 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不同的表达 在细胞凋亡中起重要作用的蛋白质，如Fas、bcl2、p53和c-myc。他们 将着眼于低氧在改变成纤维细胞中可能扮演的角色 SSC的新陈代谢。他们预计这些研究将改善 了解成纤维细胞的相互作用及其环境 总体而言，炎症状态，特别是SSC。 第一个具体目标是检查两国之间的差异 SSC和正常细胞的胶原合成是由于分化所致 CKROX的表达。稳定的转染体将用于评估cKROX为 一种转录因子。已知的调节胶原合成的因素将 评估它们对cKROX表达的影响。皮损皮肤， 非皮损皮肤、正常皮肤和成纤维细胞的克隆性群体 用凝胶位移分析、Northern印迹和RT-PCR对cKROX进行评估 表情。 在第二个具体目标中，将开发差异显示库 来自克隆的和正常的成纤维细胞，以及皮损和非皮损的成纤维细胞。 已确定的基因将被克隆和鉴定。 第三个具体目标是评估克隆的选择影响。 成纤维细胞的增殖包括细胞凋亡和缺氧。

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.