Magalhães Danielle A R, Macedo Claudia, Junta Cristina M, Mello Stephano S, Marques Márcia M C, Cardoso Renato S, Sakamoto-Hojo Elza T, Donadi Eduardo A, Passos Geraldo A S
Molecular Immunogenetics Group, Department of Genetics, Faculty of Medicine, University of São Paulo (USP), 14040-900 Ribeirão Preto, SP, Brazil.
Mol Immunol. 2005 May;42(9):1043-8. doi: 10.1016/j.molimm.2004.09.031. Epub 2004 Nov 23.
Non-manipulated inbred mouse strains constitutes an interesting model-system for in vivo studies on thymus ontogeny due to the possibility to observe the molecular events of the thymocyte maturation. In previous studies, using RT-PCR method, we have found that several immune system genes such as interleukins and MHC are differentially expressed during ontogeny of the thymus whose genes act as modulators of T-cell differentiation. To determine which other genes are modulated on a large-scale basis, we measured the levels of mRNA expression in mouse fetal thymus (14-17 days of gestation) by hybridization with cDNA microarrays containing 1,576 cDNA sequences derived from the IMAGE MTB library. T-cell maturation was monitored by detection of the T-cell receptor beta TRBV8.1-BD2.1 rearranged DNA segment. Each developmental phase of thymus, displayed a characteristic expression profile, as evaluated by the Cluster and Tree-View softwares. Genes differentially and significantly expressed were selected on the basis of significance analysis of the microarray data (SAM program). With the reclustering of only significantly expressed genes, it was possible to characterize the phases of thymus ontogeny, based on the differential profile of expression. Our method provided the detection of genes implicated in the cell signaling, such as the hematopoietic cell signal transducer gene, genes implicated in T-cell calcium influx (tyrosine phosphatase) and calcium signaling proteins (vesicle transport binding protein 3, proline rich Gla, casein kinase alpha 1 and Down syndrome homolog protein 1) and a gene important for the protein transport, including T-cell receptors chains, towards the cell membrane (Golgi SNAP receptor complex member 2). The results demonstrate that the cDNA microarray used to explore the gene expression was useful for understanding the modulation of several cell-signaling genes, including the calcium cascade pathway, which is important for individual stages of T-cell maturation and control of anergy during thymus ontogeny.
由于能够观察胸腺细胞成熟的分子事件，未经过人工处理的近交系小鼠品系构成了用于胸腺个体发育体内研究的有趣模型系统。在先前的研究中，我们使用逆转录聚合酶链反应（RT-PCR）方法发现，一些免疫系统基因，如白细胞介素和主要组织相容性复合体（MHC），在胸腺个体发育过程中差异表达，其基因作为T细胞分化的调节因子。为了确定还有哪些基因在大规模上受到调节，我们通过与包含来自IMAGE MTB文库的1576个cDNA序列的cDNA微阵列杂交，测量了小鼠胎儿胸腺（妊娠14 - 17天）中的mRNA表达水平。通过检测T细胞受体β链TRBV8.1 - BD2.1重排的DNA片段来监测T细胞成熟。胸腺的每个发育阶段都显示出一种特征性的表达谱，这是通过Cluster和Tree-View软件评估得出的。基于微阵列数据的显著性分析（SAM程序）选择差异且显著表达的基因。通过仅对显著表达的基因进行重新聚类，基于表达的差异谱来表征胸腺个体发育的阶段成为可能。我们的方法检测到了涉及细胞信号传导的基因，如造血细胞信号转导基因、涉及T细胞钙内流的基因（酪氨酸磷酸酶）和钙信号蛋白（囊泡运输结合蛋白3、富含脯氨酸的γ-羧基谷氨酸蛋白、酪蛋白激酶α1和唐氏综合征同源蛋白1）以及一个对蛋白质运输很重要的基因，包括T细胞受体链，向细胞膜运输（高尔基体可溶性NSF附着蛋白受体复合体成员2）。结果表明，用于探索基因表达的cDNA微阵列对于理解包括钙级联途径在内的几种细胞信号基因的调节是有用的，这对于T细胞成熟的各个阶段以及胸腺个体发育过程中无反应性的控制都很重要。
