Mirsepasi-Lauridsen Hengameh Chloé, Vrankx Katleen, Engberg Jørgen, Friis-Møller Alice, Brynskov Jørn, Nordgaard-Lassen Inge, Petersen Andreas Munk, Krogfelt Karen Angeliki
Department of Bacteria, Parasites and Fungi, Statens Serum Institut, Copenhagen, Denmark.
Applied Maths NV, Sint-Martens-Latem, Belgium.
Front Med (Lausanne). 2018 Nov 20;5:304. doi: 10.3389/fmed.2018.00304. eCollection 2018.
Inflammatory Bowel disease (IBD) is traditionally divided into Crohn's disease (CD) and ulcerative colitis (UC). UC is a relapsing non-transmural inflammatory disease that is restricted to the colon and is characterized by flare-ups of bloody diarrhea. CD is a chronic, segmental localized granulomatous disease that can affect any part of the entire gastrointestinal tract. Ileo-anal pouch is a procedure restoring functionality of the rectum after a colectomy. IBD is a multifactorial disease and flares of IBD are probably triggered by changes in the intestinal microbiota followed by an abnormal immune response. In this study, we aim to analyze the intestinal bacterial diversity in IBD patients during various stages of disease compared with healthy controls. Permission for human experiments and recruitment of participants was obtained from the Ethic Committee for Copenhagen County hospitals (Permission no. KA-03019, Permission no. KA-20060159). Stools from 26 healthy controls, 42 CD, 38 UC and 18 pouch patients were collected. Stool DNA extraction was performed using Qiagen, DNA mini stool kit Denmark. DGGE-PCR amplifying the V2-V3 region of 16S-rDNA gene of the bacteria was amplified by universal primers HDA1 and HDA2. Analysis of DGGE was performed blinded using BioNumerics version 7.5. After normalization, a DGGE gel band matching was performed. The similarities between profiles were calculated with a ranked Pearson correlation coefficient based on the band matching results using band intensities. Simpson's index of diversity and Pielou's species evenness were calculated. Based on the Shannon Diversity Index, UC patients had lower species diversity and bacterial evenness in comparison to healthy persons, < 0.05. However, only CD and disease pouch patients had lower species diversity compared to those with inactive disease and healthy controls. Well-functioning pouch patients had decreased species evenness in comparison to diseased pouch patients and control group. During the active disease stage in CD and pouch, the patients have a low bacterial diversity in their gut when compared to the inactive stage. In UC patients, a generally low diversity was observed at all stages of the disease compared to healthy controls.
炎症性肠病（IBD）传统上分为克罗恩病（CD）和溃疡性结肠炎（UC）。UC是一种复发性非透壁性炎症性疾病，局限于结肠，其特征为血性腹泻发作。CD是一种慢性、节段性局限性肉芽肿性疾病，可影响整个胃肠道的任何部位。回肠肛管袋术是一种在结肠切除术后恢复直肠功能的手术。IBD是一种多因素疾病，IBD发作可能由肠道微生物群变化引发，随后出现异常免疫反应。在本研究中，我们旨在分析IBD患者在疾病不同阶段与健康对照相比的肠道细菌多样性。已获得哥本哈根县医院伦理委员会的人体实验许可和参与者招募许可（许可号：KA - 03019，许可号：KA - 20060159）。收集了26名健康对照、42名CD患者、38名UC患者和18名袋术患者的粪便。使用丹麦Qiagen公司的DNA粪便微量提取试剂盒进行粪便DNA提取。通过通用引物HDA1和HDA2扩增细菌16S - rDNA基因的V2 - V3区域进行DGGE - PCR。使用BioNumerics 7.5版本进行DGGE分析时设盲。归一化后，进行DGGE凝胶条带匹配。根据条带匹配结果，使用条带强度基于排序的皮尔逊相关系数计算图谱之间的相似度。计算辛普森多样性指数和皮洛物种均匀度。基于香农多样性指数，UC患者与健康人相比物种多样性和细菌均匀度较低，P < 0.05。然而，与疾病缓解期患者和健康对照相比，只有CD患者和患病袋术患者的物种多样性较低。功能良好的袋术患者与患病袋术患者及对照组相比物种均匀度降低。在CD和袋术患者的疾病活动期，与非活动期相比，其肠道细菌多样性较低。与健康对照相比，UC患者在疾病的所有阶段普遍观察到多样性较低。
