Thymic Involution And Age-associated Changes In T Cells

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，胸腺结构发生了深刻的变化，胸腺经历了一个逐渐缩小的过程。在这种下降之后，循环中的幼稚T细胞和细胞介导的免疫反应的数量减少，这可能在老化宿主内观察到的肿瘤发生、自身免疫和感染性疾病的增加中发挥作用。尽管对胸腺老化的病理生理学进行了广泛的研究，但涉及胸腺退化过程的确切分子机制仍不清楚。为了描绘衰老胸腺内发生的分子变化，使用从不同年龄的小鼠胸腺分离得到的RNA进行微阵列分析。以2、4、6、12和18月龄BALB/c小鼠胸腺提取的mRNA为材料，采用本实验室研制的三种不同定制的基因芯片进行基因芯片分析。对于每个阵列，每个实验使用每个年龄组的三个不同的放射性标记的cDNA进行三次杂交，每个RNA产生大约6个基因复制。在分析之后，然后使用计算机程序CLUSTER进行分级聚类分析以确定不同年龄组之间的基因表达变化和共表达的程度。我们的结果表明，与2月龄相比，大约100个基因在12和18个月龄胸腺中的表达显著上调，而在4个月和6月龄胸腺中的表达没有明显上调。比较年长的胸腺和年轻的胸腺，可以观察到显著的基因表达上调。与G蛋白偶联受体、转录因子、激素受体、T细胞信号、迁移、造血和细胞周期相关的基因表达在12个月和18个月龄胸腺中被发现增加。这一表述的意义还有待确定。我们初步选择了CCR5、LIF、TSHR、IGF-2R和BDNF等5个上调基因进行验证和功能分析。这些基因中的每一个都已被证明存在于胸腺组织中；然而，它们在胸腺功能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老化细胞的分子图谱的可靠性，这些来源包括培养的和新鲜分离的老化细胞。第一个里程碑将是确定与胸腺退化相关的基因的特征和选择。我们计划对不同年龄、H-2和遗传背景的小鼠胸腺和脾中的基因表达进行一系列分析，以及已知的退化小鼠模型。我们目前的数据表明，胸腺退化可能是依赖于应变的，可能部分与不同的遗传因素有关，而不是简单的衰老。我们设计了一个由大约5,000个已知的小鼠克隆组成的特定的小鼠cDNA阵列，以便于进行这一分析。新的定制过滤器将允许我们在每次分析中使用更少的材料，因此我们可以分析单个老鼠而不是池，以克服每次分析需要大量组织的问题。一旦建立了每个菌株和年龄的可靠基因图谱，我们将识别和定义仅在老年胸腺细胞和老年外周淋巴或T细胞群体中表达的基因。目前尚不清楚某些淋巴器官或细胞成分是否对长寿和寿命起着关键作用。年轻和老年胸腺细胞、胸腺CD4和CD8亚群、胸腺哺育细胞、胸腺上皮、脾和外周脾T细胞亚群也正在分离和研究中，以期从老年小鼠的胸腺细胞亚群和外周T细胞中识别和区分候选基因。

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 labortory. For each array, three hybridizations using three different radiolabelled 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. The significance of this expression remains to be determined. We have initially selected five upregulated genes for verification and functional analysis including CCR5, LIF, TSHR, IGF-2R and BDNF. Each of these genes has been shown to be present in thymic tissue; however, their precise role in thymic function 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. The new custom-made filters will allow us to utilize less material for each analysis so we can analyze individual mice instead of pools to overcome the requirement of large amount of tissues for each analysis. Once reliable gene profiles are established per strain and age, we shall identify and define the genes expressed only in aged thymic cells vs. aged peripheral lymphoid or T cell populations. It is unclear whether certain lymphoid organs or cellular components play a critical role in longevity and lifespan. Young and old thymocytes, thymic CD4 and CD8 subsets, thymic nurse cells, thymic epithelium, spleen, and peripheral splenic T cell subsets are also currently being isolated and studied with the hope to identify and distinguish candidate genes from the thymic cell subsets and peripheral T-cells of aged mice.