DNA array analysis of interleukin-2-regulated immediate/early genes.
DNA array analysis of interleukin-2-regulated immediate/early genes.
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DOI：
10.1186/1476-9433-1-2
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发表时间：
2002-11-18
期刊：
影响因子：
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通讯作者：
Smith, Kendall A
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文献类型：
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作者：
Beadling, Carol;Smith, Kendall A
淋巴细胞活化在胚细胞发生、细胞周期进展、DNA复制和有丝分裂中达到高潮。这些复杂的细胞变化几乎同时由多个配体和受体编程，这些配体和受体触发特定的信号转导途径和转录因子。到目前为止，发现由每个配体/受体对调控的基因一直受到现有技术的阻碍。为了鉴定白细胞介素-2（IL-2）响应基因，用抗CD 3预激活人外周血单核细胞（PBMC），静置，并用IL-2重新刺激4小时。使用Affymetrix U95 Av 2寡核苷酸阵列分析基因表达。为了确定将基因评分为真正的IL-2靶标的最严格参数，首先检查了19个已知的IL-2调节基因的表达。所有均诱导至少2倍，在p < 0.05时荧光强度差异≥ 100。另外53个独特的基因符合这些标准。为了确定哪些是T细胞中的立即/早期IL-2靶标，在放线菌酮存在下用IL-2刺激纯化的T细胞4小时以防止次级基因表达。在PBMC中鉴定的72个基因中，20个在纯化的T细胞中被检测为立即/早期IL-2调节的基因。此外，27个独特的基因是IL-2调节的T细胞，但不是在PBMC中。对于一个成功的还原论的方法来分析淋巴细胞活化中的基因表达，有必要检查纯化的细胞群和立即/早期基因表达所涉及的每个配体/受体对的调节。这种方法应该允许发现基因调控的所有参与淋巴细胞活化的配体/受体对。
Lymphocyte activation culminates in blastogenesis, cell cycle progression, DNA replication and mitosis. These complex cellular changes are programmed almost simultaneously by multiple ligands and receptors that trigger specific signal transduction pathways and transcription factors. Until now, the discovery of the genes regulated by each ligand/receptor pair has been hampered by the technologies available. To identify interleukin-2 (IL-2)-responsive genes, human peripheral blood mononuclear cells (PBMC) were pre-activated with anti-CD3, rested, and restimulated with IL-2 for 4 hr. Gene expression was analyzed using Affymetrix U95Av2 oligonucleotide arrays. To determine the most stringent parameters to score a gene as a bona fide IL-2 target, the expression of 19 known IL-2-regulated genes was examined first. All were induced at least 2-fold, with a difference in fluorescent intensity of ≥ 100 at p < 0.05. An additional 53 unique genes met these criteria. To determine which of these were immediate/early IL-2 targets in T cells, purified T cells were stimulated with IL-2 for 4 hr in the presence of cycloheximide to prevent secondary gene expression. Of the 72 genes identified in PBMCs, 20 were detected as immediate/early IL-2-regulated genes in purified T cells. In addition, 27 unique genes were IL-2-regulated in T cells but not in PBMCs. For a successful reductionist approach to the analysis of gene expression in lymphocyte activation, it is necessary to examine purified cell populations and immediate/early gene expression regulated by each ligand/receptor pair involved. This approach should allow the discovery of genes regulated by all of the ligand/receptor pairs involved in lymphocyte activation.