Mechanisms that Regulate Thymic Involution and Age-Assoc

免疫系统老化的后果之一是胸腺退化的过程。由于胸腺上皮萎缩和胸腺生成减少，胸腺的结构发生了深刻的变化，导致胸腺体积逐渐缩小。这种下降是系统性的，随后是循环幼稚T细胞和细胞介导的免疫应答的数量减少，这可能在衰老宿主内观察到的肿瘤发生、自身免疫和感染性疾病增加中发挥作用。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。为了描述衰老胸腺内发生的分子变化，使用从不同年龄小鼠的胸腺中分离的RNA进行微阵列分析。使用来自胸腺的mRNA的2，4，6，12和18个月大的BALB/c小鼠;微阵列分析进行使用三个不同的定制的cDNA微阵列在我们的实验室内开发。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产生约6个基因重复。分析后，使用计算机程序Cluster进行分层聚类分析，以确定不同年龄组之间基因表达变化和共表达的程度。我们的研究结果表明，与2月龄胸腺相比，12月龄和18月龄胸腺中约100个基因的表达显著上调，而4月龄和6月龄胸腺中的表达则没有显著上调。与年轻组相比，老年胸腺的基因表达显著上调。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在12个月和18个月大的胸腺中被发现增加。我们初步选择了5个上调基因进行验证和功能分析，包括CCR 5，LIF，TSHR，IGF-2 R和BDNF。此后，我们已经验证了胸腺组织中这些基因的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来自确定的老化来源的分子谱老化细胞的可靠性，包括来自培养物和新鲜分离的老化细胞。第一个里程碑将是胸腺退化相关基因的明确表征和选择。我们计划对不同年龄、H-2和遗传背景以及已知退化小鼠模型的小鼠胸腺和脾脏中的基因表达进行一系列分析。我们目前的数据表明，胸腺退化可能是应变依赖性的，可能部分与不同的遗传因素，而不是简单的老化。我们设计了一个特定的小鼠cDNA阵列，由大约5,000个已知的小鼠cDNA克隆组成，以促进这种分析。我们目前正在分析从老年脾脏、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。比较只在老年胸腺细胞和老年外周淋巴器官或淋巴细胞中表达的基因。目前还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。年轻和年老的胸腺CD 4和CD 8亚群，胸腺护士细胞，胸腺上皮细胞和胸腺和外周脂肪细胞目前也正在分离和研究，希望能识别和区分候选基因，可能发挥作用的胸腺退化过程中，以及与年龄相关的细胞信号和活动的变化相关的基因。 总体而言，该项目的主要目标是在衰老过程中产生胸腺，脾脏和淋巴结中的全面基因表达谱，以确定独特和共同的基因以及在这些不同器官系统中以年龄依赖性方式表达的功能相关基因组。我们最初将我们的努力集中在胸腺上，因为它的退化被认为是解决与衰老相关的免疫缺陷的最重要的障碍之一。

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.