cDNA芯片技术筛选斑马鱼皮肤免疫相关差异表达基因

摘要

The objective of this study is to screen the differential gene expression in response to a pathogenic infection in the skin of fish, and to provide a basis for understanding the fish skin immune mechanism at the molecular level. The immune response in the skin was analyzed by using Staphylococcus-induced zebrafish as a model, and applying the affymetrix zebrafish cDNA microarray hybridized to the skin tissues. Total RNAs were isolated from the skin tissues of adult fish, labeled with biotin, and hybridized to zebrafish cDNA gene chips. The expression profiles from the hybridization to 15 617 genes in the zebrafish cDNA array were analyzed by the Ge-neChip Operating Software (GCOS1.4). Out of 15 617 genes in zebrafish cDNA chips, a total of 175 was identified to be significantly expressed in the skin tissues, of which 150 were up-regulated and 25 were down-regulated. Among the 150 up-regulated genes, the functions of 91 genes were known and 59 were unknown. Furthermore, by using the Gene Ontology (GO) method the differential expression genes could be categorized into 13 functional groups, and some of them are considered as potential candidates in the fish skin immune response. Thus, several genes related to immune response in the skin were identified, including major histocompatibility complex class I genes (UEA, UFA), complement component (Clq, C7-1), lectin (HBL3, LGALS1L3), early growth response 1 (EGR-1), tumor necrosis factor superfamily (TNFSF10L4), coagulation factor V(F5), transferrin-a (TF-a), and several proteases. The Affymetrix zebrafish cDNA microarray is useful for identifying the functional genes involved in the skin immune defense of fish: that is, MHC I, complement, lectin, and proteases. However, future analysis of the function of these genes may contribute to an understanding of the mechanisms of Staphylococcus pathogenesis and mucosal bacterial interactions.%研究旨在筛选与鱼类皮肤免疫相关的功能基因,试图解释鱼类皮肤局部免疫应答的分子机制.采用斑马鱼(Danio rerio)基因cDNA芯片(affymetrix),以葡萄球菌(Staphylococcus sp.)感染诱导的成体斑马鱼为实验动物模型,从斑马鱼皮肤组织中提取总RNA,经Biotin荧光标记与拥有15 617个cDNA片段的基因芯片杂交,对斑马鱼皮肤组织中基因表达谱进行初步分析.在斑马鱼皮肤组织中共检测出175个差异表达基因,其中有150个上调表达基因(ratio＞2.0)和25个下调表达基因(ratio＞0.5);在皮肤组织150个上调基因中,91个为已知功能基因,59个为未知功能基因.根据基因文库同源功能基因(GO)分析,将175个差异表达基因分为13个主要的生物功能与代谢通路,其中参与免疫应答相关基因包括鱼主要组织相容性复合体(MHC)I类基因区的基因(UEA,UFA)、补体(Clq,C7-1)、凝集素(HBL3,LGALS1L3)、应急反应生长基因(EGR-1)、肿瘤坏死因子超家族基因(TNFSF10L4)、凝血因子(F5)、转铁蛋白基因(TF-α)和一些蛋白酶等.结果证实,MHC I类分子、补体、凝集素和蛋白酶等参与鱼类皮肤的抗葡萄球菌感染免疫应答,为将来以斑马鱼感染模型研究皮肤局部与细菌的互作机制提供了科学参考；同时表明采用基因芯片方法初探斑马鱼皮肤免疫系统是基本可行的.