Mechanisms that Regulate Thymic Involution and Age-Assoc

调节胸腺复旧和年龄相关的机制

• 批准号: 6674124
• 负责人: DENNIS D. TAUB
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6674124
• 关键词: T lymphocyte; aging; atrophy; developmental immunology; functional /structural genomics; gene expression; genetic strain; laboratory mouse; spleen; thymus; tissue /cell culture

One of the consequences of an aging immune system is the process of thymic involution. The thymus undergoes a progressive reduction in size due to profound changes in its architecture associated with thymic epithelia atrophy and decreased thymopoiesis. This decline is systemically followed by decreased numbers of circulating naive T cells and cell-mediated immune responses which may play a role in the increased tumorigenesis, autoimmunity, and infectious diseases observed within an aging host. Despite the extensive study of the pathophysiology of the aging thymus, the precise molecular mechanism involved in the involution process remains unclear. In an effort to profile molecular changes that occur within the aging thymus, microarray analysis was performed using RNA derived from thymus isolated from mice of varying ages. Using mRNA derived from the thymi of 2, 4, 6, 12 and 18 month old BALB/c mice; microarray analysis was performed using three distinct custom-made cDNA microarrays developed within our laboratory. For each array, three hybridizations using three different radiolabeled cDNAs of each age group were performed per experiment yielding approximately 6 gene replicates per RNA. After analysis, hierarchical cluster analysis was then performed to determine the degree of gene expression changes and co-expression between the different age groups using the computer program, Cluster. Our results have demonstrated that the expression of approximately 100 genes were significantly up-regulated in 12 and 18 but not in 4 and 6 month old thymus compared with 2 month old thymus. Significant upregulation in gene expression was observed upon comparing older thymi with the younger groups. Gene expression associated with G protein-coupled receptors, transcription factors, hormone receptors, T cell signaling, migration, hematopoesis, and cell cycle were found to be increased in the 12- and 18-month old thymi. We have initially selected five upregulated genes for verification and functional analysis including CCR5, LIF, TSHR, IGF-2R and BDNF. We have since verified the patterns of mRNA and protein expression for each of these genes within the thymic tissue. However, the precise role of these genes in thymic function and involution remains to be defined. The success of this project relies upon the reliability of the molecular profiling aged cells from defined aged sources, both from culture and freshly isolated aged cells. The first milestone will be the definitive characterization and selection of genes associated with thymic involution. We plan to conduct a series analysis of gene expression in the thymi and spleens of mice of varying ages, H-2 and genetic backgrounds, and known involution mouse models. Our current data would suggest that thymic involution may be strain dependent and may in part be associated with distinct genetic factors rather than simply aging. We have designed a specific murine cDNA array consisting of approximately 5,000 known mouse cDNA clones to facilitate of this analysis. We are currently analyzing the data obtained from the gene profiles of aged spleens, thymi, bone marrow, B cells, T cells and thymocytes. Comparisons are being made between genes expressed only in aged thymic cells vs. aged peripheral lymphoid organs or lymphocytes. It is unclear whether certain lymphoid organs or cellular components play a critical role in longevity and lifespan. Young and old thymic CD4 and CD8 subsets, thymic nurse cells, thymic epithelium and thymic and peripheral adiopocytes are also currently being isolated and studied with the hope to identify and distinguish candidate genes that may play a role in the thymic involution process as well as those genes associated with age-related alterations in cell signaling and activity. Overall, the major goal of this project is to produce a comprehensive gene expression profile in the thymus, spleen, and lymph nodes during the aging process to identify unique and common genes and functionally related groups of genes that are expressed in age-dependent manner in these different organ systems. We have initially focused our efforts on the thymus, as its involution is believed to be one of the most significant obstacles to overcome in addressing the immunological deficits associated with aging.

免疫系统老化的后果之一是胸腺退化过程。由于与胸腺上皮萎缩和胸腺生成减少相关的结构的深刻变化，胸腺的尺寸逐渐减小。这种下降系统性地伴随着循环初始 T 细胞数量和细胞介导的免疫反应的减少，这可能在衰老宿主体内观察到的肿瘤发生、自身免疫和传染病增加中发挥作用。尽管对衰老胸腺的病理生理学进行了广泛的研究，但参与退化过程的精确分子机制仍不清楚。为了分析衰老胸腺内发生的分子变化，使用从不同年龄小鼠分离的胸腺中提取的 RNA 进行了微阵列分析。使用源自 2、4、6、12 和 18 月龄 BALB/c 小鼠胸腺的 mRNA；使用我们实验室开发的三种不同的定制 cDNA 微阵列进行微阵列分析。对于每个阵列，每个实验使用每个年龄组的三种不同放射性标记的cDNA进行三次杂交，每个RNA产生大约6个基因复制。分析后，使用计算机程序Cluster进行层次聚类分析，以确定不同年龄组之间基因表达变化和共表达的程度。我们的结果表明，与 2 月龄胸腺相比，大约 100 个基因的表达在 12 月龄和 18 月龄胸腺中显着上调，但在 4 月龄和 6 月龄胸腺中没有显着上调。比较老年胸腺与年轻群体时观察到基因表达显着上调。研究发现，12 个月和 18 个月大的胸腺中与 G 蛋白偶联受体、转录因子、激素受体、T 细胞信号传导、迁移、造血和细胞周期相关的基因表达有所增加。我们初步选择了5个上调基因进行验证和功能分析，包括CCR5、LIF、TSHR、IGF-2R和BDNF。此后，我们验证了胸腺组织内每个基因的 mRNA 和蛋白质表达模式。然而，这些基因在胸腺功能和退化中的确切作用仍有待确定。 该项目的成功依赖于对来自指定老化来源（培养物和新鲜分离的老化细胞）的老化细胞进行分子分析的可靠性。第一个里程碑将是与胸腺退化相关的基因的明确表征和选择。我们计划对不同年龄、H-2 和遗传背景的小鼠胸腺和脾脏以及已知的退化小鼠模型的基因表达进行一系列分析。我们目前的数据表明，胸腺退化可能是菌株依赖性的，并且可能部分与不同的遗传因素有关，而不仅仅是与衰老有关。我们设计了一个特定的小鼠 cDNA 阵列，其中包含大约 5,000 个已知的小鼠 cDNA 克隆，以方便进行此分析。我们目前正在分析从衰老的脾脏、胸腺、骨髓、B 细胞、T 细胞和胸腺细胞的基因谱中获得的数据。目前正在对仅在老化胸腺细胞中表达的基因与老化外周淋巴器官或淋巴细胞中表达的基因进行比较。目前尚不清楚某些淋巴器官或细胞成分是否在长寿和寿命中发挥关键作用。目前也正在分离和研究年轻和年老的胸腺CD4和CD8亚群、胸腺护理细胞、胸腺上皮以及胸腺和外周脂肪细胞，希望能够识别和区分可能在胸腺退化过程中发挥作用的候选基因，以及与年龄相关的细胞信号传导和活性改变相关的基因。 总体而言，该项目的主要目标是在衰老过程中产生胸腺、脾脏和淋巴结的全面基因表达谱，以识别在这些不同器官系统中以年龄依赖性方式表达的独特和常见基因以及功能相关的基因组。我们最初将工作重点放在胸腺上，因为胸腺的退化被认为是解决与衰老相关的免疫缺陷问题时需要克服的最重要的障碍之一。