应用基因表达谱芯片研究内毒素诱导的人树突细胞成熟前后免疫相关基因的差异表达

Screening the differentially expressed genes of immature and LPS-induced mature dendritic cells using cDNA microarray

目的研究人外周血来源的树突细胞(DCs)经内毒素诱导成熟过程中基因表达谱的改变。方法采用GM-CSF及IL-4诱导人外周血单核细胞获得非成熟DCs,以100ng/ml内毒素(LPS)作用48h获得成熟DCs,收集细胞提取总RNA,应用SuperArray低通量全长基因cDNA芯片筛选DCs成熟前后差异表达的基因。结果经LPS诱导后DCs高表达成熟DCs表面标志物。经LPS处理48h后诱导成熟的DCs与未经处理的非成熟DCs相比,共有50条基因出现差异表达,占芯片基因总数的30.3%。其中35条基因表达增加,15条基因表达下降。细胞因子/受体和趋化因子/受体基因中有12条表达上调,2条表达下调,抗原获取及提呈相关分子中有10条表达上调,5条表达下调,膜受体及信号转导分子中13条表达上调,8条表达下调。RT-PCR进一步证实了基因表达谱芯片的结果。结论LPS对DCs成熟过程中的基因表达具有显著影响,其范围涉及细胞因子、趋化因子及受体,抗原提呈相关分子及细胞内信号转导分子等。分析这些差异基因的表达,对进一步全面了解DCs成熟过程有重要意义。

Objective To study the changes in gene expression profiles of dendritic cells （DCs） during their maturation processes using cDNA rnicroarray. Method DCs were generated from human peripheral blood monocytes induced by GM-CSF and IL-4, and lipopolysaccharide （LPS） was used to induce the maturation of DCs. After inducing for 48h, cells were collected to extract the general RNA. Genes expressions of mature DCs （maDC） and immature DCs （imDC） were analyzed using cDNA microarray, and immature DCs without induction served as control. Results DCs expressed highly surface markers of mature DCs after LPS induction. The studied data showed that among those 165 target genes, a total of 50 genes （30. 3%） exhibited differential levels of expression in LPS-induced mature DCs. Thirty-five genes were up-regulated and fifteen genes were down-regulated. Maturation-dependent up-regulation, defined by a differential expression ratio of ≥2, included twelve cytokine and chemokine genes, ten antigen uptake and presentation genes and thirteen signal transduction molecule genes. Reciprocally, maturation-dependent down-regulation, defined by a differential expression ratio of 40. 5, occurred with two cytokine, five antigen uptake and presentation genes and eight signal transduetion molecule genes. Semi-quantitative reverse transcription polymerase chain reaction （RT-PCR） was employed to further confirm the results of cDNA microarray. Conclusion It is suggested that LPS has significant influence on many genes of DCs in their maturing processes. Those changed genes include cytokines, chemo- kines and their receptors, antigen uptake and presentation molecules and signal transduction molecules. Analysis on these differentially expressed genes is helpful in understanding the maturing processes of DCs.