Molecular cloning and functional analysis of genes associated with mucosal defense to investigate patbophysiology of inflammatory bowel disease

我们鉴定了抵抗素样分子β(RELm-β)和缺失的恶性脑肿瘤1(DMBT1)是细菌攻击无菌小鼠诱导的与粘膜防御肠道菌群相关的分子。我们研究了这些基因和蛋白在实验性和人类炎症性肠病黏膜组织中的表达及其分子的诱导机制。RELm-β-β在结肠中选择性地表达，并从上皮细胞分泌到肠腔内。在粪便中存在免疫反应的RELm-β，提示该蛋白可能与肠道菌群相互作用。我们通过CFU实验研究了RELm-β是否能够抑制肠道菌群的细菌生长，结果表明该蛋白具有抗金黄色葡萄球菌的能力。该基因在DSS-结肠炎中也被诱导，即使在通过去除DSS而从活动性炎症中恢复之后。Northern blotting显示，人的同源物DMBT1的大小为7kb，在对照组中弱表达，但在三分之二的IBD分离株中显著诱导表达。在三分之一的IBD分离株中观察到额外的6.5kb条带。实时荧光定量RT-PCR结果显示，DC和CD中DMBT1基因的表达水平均显著高于CD。但免疫组织化学检测未发现DMBT1蛋白表达存在质的差异。该基因是由IL-1β等可溶性因子诱导的。

We identified resistin-like molecule β (RELM-β) and deleted malignant brain tumors 1(DMBT1) as molecules induced by bacterial challenge to germ-free mice and associated with mucosal defense against enteric flora. We investigated expression of those genes and proteins in mucosal tissues of experimental and human inflammatory bowel disease and induction mechanisms of the molecules.Antibacterial activity of RELM-βRELM-β was selectively expressed in the colon and secreted from epithelial cells into the lumen. Immunoreactive RELM-β was present in stool, suggesting possible interaction of the protein with enteric flora. We investigated whether or not RELM-β is capable of inhibiting bacterial growth against panels of enteric flora by CFU assay, resulting that this protein exhibited anti-bacterial capacity against Staphylococcus aureus.Epithelial induction of a human homologue of murine CRP-ductin, Deleted in Malignant Brain Tumors 1 (DMBT1)Induction of CRP-ductin mRNA was initially detected by cDNA microarray. This gene was also induced in DSS-colitis even after recovery from active inflammation by the removal of DSS. Northern blotting revealed that the human homologue, DMBT1 with 7 kb in size, was weakly expressed in control but remarkably induced in two-thirds of IBD isolates. An additional 6.5 kb band was noticed in one-third of IBD isolates. Real-time RT-PCR demonstrated that DMBT1 mRNA level was significantly higher in both DC and CD. However, qualitative difference in DMBT1 protein expression was not identified by immunohistochemistry. This gene was induced by soluble factors including IL-1β.