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.

我们将抗素样分子β（RERM-β）和删除的恶性脑肿瘤1（DMBT1）鉴定为细菌对无细菌小鼠挑战的分子，并与针对肠菌群的粘膜防御有关。我们研究了这些基因和蛋白质在实验和人类炎症性肠病的粘膜组织中的表达以及分子的诱导机制。选择RELM-βRelm-β的抗细菌活性以在结肠中表达并从上皮细胞中分泌到流明中。免疫反应性RERM-β存在于粪便中，表明蛋白质与肠菌群可能相互作用。我们研究了通过CFU测定法对肠菌群抑制细菌的生长是否能够通过CFU测定抑制细菌的生长，从而导致这种蛋白暴露于金黄色牙菌的抗菌能力。 cDNA微阵列。即使通过去除DSS从主动感染中恢复活性后，该基因也被诱导。 Northern印迹显示，人类同源物大小为7 kb的DMBT1在对照中弱表达，但在三分之二的IBD分离株中非常诱导。在三分之一的IBD分离株中，还记录了另外6.5 kb的频带。实时RT-PCR证明DMBT1 mRNA水平在DC和CD中均明显更高。但是，未通过免疫组织化学鉴定DMBT1蛋白表达的质量差异。该基因是由包括IL-1β在内的固体因子诱导的。

项目成果

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）。

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）。

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》。

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。

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)。