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

粘膜防御相关基因的分子克隆和功能分析，以研究炎症性肠病的病理生理学

• 批准号: 13470249
• 负责人: FUKUSHIMA Kouhei
• 金额: $ 10.43万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470249/
• 关键词: intestinal flora; inflammatory bowel disease; microarray; epithelial cells; Germ-free; ulcerative colitis; Crohn's disease; resistin-like molecule; FIZZ; クローニング

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.

我们发现抵抗素样分子β (RELM-β)和删除恶性脑肿瘤1（DMBT1）是细菌攻击无菌小鼠诱导的分子，并与肠道菌群的粘膜防御有关。我们研究了这些基因和蛋白在实验性和人类炎症性肠病粘膜组织中的表达及其诱导机制。RELM-β的抑菌活性在结肠中选择性表达，并由上皮细胞分泌到管腔中。粪便中存在免疫反应性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是否能够抑制细菌对肠道菌群的生长，结果表明该蛋白对金黄色葡萄球菌具有抗菌能力。在恶性脑肿瘤中缺失的小鼠CRP-ductin 1 （DMBT1）的人同源物的上皮诱导作用通过cDNA芯片初步检测了CRP-ductin mRNA的诱导作用。该基因在DSS-结肠炎中也被诱导，甚至在移除DSS后活动性炎症恢复后也是如此。Northern blotting显示，人类同源物DMBT1大小为7kb，在对照中弱表达，但在三分之二的IBD分离株中显著诱导。在三分之一的IBD分离株中发现了额外的6.5 kb条带。Real-time RT-PCR结果显示，DC和CD的DMBT1 mRNA水平均显著升高，但免疫组化未发现DMBT1蛋白表达的质的差异。该基因由IL-1β等可溶性因子诱导。

项目成果

K Fukushima ほか: "Paradoxical decrease of mitochondrial DNA deletion in epithelial cell of active ulcerative colitis patients"Am J Physiol(Gastrointestinal Liver Physiol). (In press). (2004)

K Fukushima 等人：“活动性溃疡性结肠炎患者上皮细胞中线粒体 DNA 缺失的矛盾性减少”Am J Physiol（胃肠肝脏生理学）（2004 年出版）。

福島浩平: "Inflammatory Bowel Disease-associated gene expression in intestinal epithelial cells by differential cDNA screening and mRNA display"Inflammatory Bowel Disease. (in press). (2003)

Kohei Fukushima：“通过差异 cDNA 筛选和 mRNA 展示肠上皮细胞中炎症性肠病相关基因的表达”炎症性肠病（印刷中）。

Fukushima K, et al.: "Decreased expression of syncollin mRNA in colonic epithelial cells of bacteria-challenged germ-free mice and ulcerative colitis."Digestive Diseases and Sciences. (In press). (2004)

Fukushima K 等人：“受细菌攻击的无菌小鼠和溃疡性结肠炎的结肠上皮细胞中 Syncollin mRNA 的表达降低。”消化疾病与科学。

K.Fukushima et al.: "Epithelial induction of serum amyloid A in expenmental mucosal inflammation"Digestive Diseases and Sciences. (in press).

K.Fukushima 等人：“实验性粘膜炎症中血清淀粉样蛋白 A 的上皮诱导”《消化疾病与科学》。

福島浩平 ほか: "消化管(IV)炎症性腸疾患"臨床透析. 19. 934-939 (2003)

Kohei Fukushima 等人：“胃肠道 (IV) 炎症性肠病”临床透析 19. 934-939 (2003)。