【作者】 崔潇；

【导师】 左秀丽；

【作者基本信息】 山东大学 ， 内科学（消化系病）， 2020， 硕士

【摘要】 研究背景和目的炎症性肠病(inflammatory bowel disease,IBD)包括溃疡性结肠炎(Ulcerative colitis,UC)和克罗恩病(Crohn disease,CD),是一种反复发作的肠道炎症性疾病,致病机制不明。近期研究表明,其病因可能是多因素的自发性炎症,由肠道对微生物的免疫引起的。通过16S-rRNA基因测序的方法可揭示UC患者的肠道菌群特征,然而,肠道微生物变化与疾病进程之间的相关性仍然不清楚。而UC的疾病进程与疾病严重程度的评估有关,并且反复发作对病人生活质量造成严重的负面影响,甚至增大了结直肠癌的发作风险。肠道需要复杂且高度调节的肠道免疫反应在免疫耐受和免疫反应中取得平衡,研究表明UC伴有多种免疫细胞功能失调以及相关细胞因子调节失衡。树突状细胞(dendriticcell,DC)存在于人体的大多数组织中,识别抗原,在T细胞的分化中起着举足轻重的免疫枢纽作用。表达于免疫细胞表面的免疫检查点分子如 T 细胞免疫球蛋白黏蛋白 3(T cell immunoglobulin and mucin-domain containing molecule 3,Tim3),参与调节局部微环境,是免疫耐受的调节剂。然而Tim3在肠炎不同阶段以对DC的作用机制鲜有报道。益生菌作为辅助治疗已应用于UC,罗伊氏乳杆菌(Lactobacillus reuteri,L.reuteri)和酪酸梭菌(Clostridium butyricum.C.butyricum)可抑制促炎性细胞因子的产生。但是在肠炎不同阶段中,益生菌对固有免疫的调节作用鲜有报道。我们假设疾病发展过程中可能存在一些特殊肠道菌群特征,并且对益生菌的干预治疗有指导作用。为了验证这一假设,我们调查了健康志愿者和UC患者的肠道菌群变化,以及益生菌对小鼠肠炎不同阶段的固有免疫的调节机制。研究内容第一部分1.患者与健康对照的粪便样本大体观招募临床溃疡性结肠炎病人和健康志愿者,收集粪便,利用高通量测序技术基于细菌16S-rDNA基因序列对IBD患者粪便细菌进行生物多样性与结构组成分析。2.疾病进程对肠道菌群的影响将UC患者分为根据患者的状况根据患病时长,分为短期病程(SC:疾病病程为1-2年),中等病程(MC:疾病病程为3-9年)和长期病程(LC:病程大于或等于10年)三类。探究粪便菌群多样性以及细菌群落组成有无差异第二部分1.急性肠炎的固有免疫状态以及益生菌的干预影响使用DSS急性肠炎模型小鼠,进行罗伊氏乳杆菌灌胃干预,研究益生菌干预对肠道DC比例及亚群的变化以及Tim3的表达差异研究。2.益生菌及其代谢产物对低度肠炎的固有免疫影响使用TNBS低度肠炎模型小鼠,进行酪酸梭菌及其代谢产物丁酸盐干预,研究肠道DC比例及亚群的变化,Tim3的表达差异,并与急性肠炎对比。研究结论第一部分1.UC粪便菌群生物多样性增加。UC组与HC组相比,变形菌门和放线菌门的比例降低,而厚壁菌门比例明显增加。2.有10年以上病程的患者的厚壁菌门比例没有明显减少。其中Bifidobacteriumh和Blautia仅在疾病的早期才发生显着变化。在病程超过十年的疾病组中,Enterococcus明显高于短期疾病和正常对照。第二部分1.DSS诱导结肠炎小鼠结肠中CD11c+CD11b+DCs和CD11c+103+DCs的表达明显低于正常对照小鼠(P<0.05),而罗伊氏乳杆菌的干预使CD11c+CD1 1b+DCs和CD11c+103+DCs的比例显著增高,这些结果表明罗伊氏乳杆菌可激活致耐受性DC的保护性作用,树突状细胞参与并减少了 DSS诱导的肠道炎性免疫反应。2.TNBS低度炎症的肠道病理结果无明显改变,但炎症细胞因子仍有增加,酪酸梭菌和丁酸盐可降低IL-17A的表达,也使低度炎症模型小鼠脾脏CD1 1c+CD11b+DC细胞IL-10的分泌显著增加。研究意义与不足这项研究首次将UC的肠道菌群变化与疾病的持续时间结合起来,可能有助于指导UC的分期。探究不同时期益生菌干预对肠道固有免疫状态的影响,指导个体化治疗。该课题不足之处是益生菌对树突状细胞的具体作用途径以及干预后对整体肠道菌群的影响仍需要进一步探讨。更多还原

【Abstract】 Background and AimsInflammatory bowel disease(IBD)includes ulcerative colitis(UC)and Crohn disease(CD).It is a recurrent intestinal inflammatory disease with unknown pathogenesis.Recent studies have shown that the cause may be spontaneous inflammation of multiple factors,such as intestinal immunity to microorganisms.The method of 16S-rRNA gene sequencing can reveal the characteristics of intestinal flora in patients with UC.However,the correlation between intestinal microbial changes and disease progression is still unclear.The course of UC disease is related to the assessment of the severity of the disease,having a serious negative impact on the quality of patients’ life,and even increase the risk of colorectal cancer.The intestine requires a complex and highly regulated intestinal immune response to achieve a balance between immune tolerance and immune response.Studies have shown that UC is accompanied by a variety of immune cell dysfunction and related cytokine regulation imbalance.Dendritic cells(DCs)are present in most tissues of the body,recognizing antigens,and play a pivotal role as an immune hub in the differentiation of T cells.Immune checkpoint molecules expressed on the surface of immune cells,such as T cell immunoglobulin and mucin-domain containing molecule 3(Tim3),participating in the regulation of the local microenvironment and are regulators of immune tolerance.However,Tim3’s mechanism of action on DC in different stages of enteritis is rarely reported.Probiotics have been used as adjuvant therapy in UC,Lactobacillus reuteri(L reuteri)and Clostridium butyricum(C.butyricum)can inhibit the production of pro-inflammatory cytokines.However,in different processes of enteritis,the regulation of probiotics on innate immunity is rarely reported.We hypothesized that there may be some special intestinal flora characteristics while disease development,and it has a guiding role in the intervention treatment of probiotics.To test this hypothesis,we investigated the changes in the intestinal flora of healthy volunteers and UC patients,as well as the mechanisms by which probiotics regulate the innate immunity of mice at different stages of enteritisMethodPart 11.The general view of stool samples from patients and healthy controlsRecruit clinical UC patients and healthy volunteers to collect feces,and analyze the diversity of microbiota and structural composition of fecal bacteria in IBD patients based on the 16S-rDNA high-throughput sequencing technology.2.The impact of disease progression on intestinal floraUC patients are divided into short-term disease course(SC:disease course is 1-2 years),medium-term disease course(MC:disease course is 3-9 years)and long-term disease course(LC:disease course is greater than or equal to 10 years)three categories Investigate whether the fecal flora diversity and bacterial community composition are differentPart 21.The innate immune status of acute enteritis and the influence of probiotic interventionTo study the changes of the proportion and subgroups of intestinal DC and the expression difference of Tim3 in DSS acute enteritis model mice by Lactobacillus reuteri probiotic intervention.2.The innate immune effects of probiotics and the metabolites on low-grade enteritisUsing TNBS model mice with low-grade enteritis,intervention with C.butyricum and its metabolite butyrate to study changes in intestinal DC ratio and subgroups.Figure the differences in expression of Tim3 and compare with acute enteritis ResultsPart 11 The diversity of UC fecal flora is increased.Compared with the HC group,the levels of Proteobacteria and Actinobacteria decreased in the UC group,while Firmicutes increased significantly.2 The number of Firmicutes bacteria in patients with a disease course of more than 10 years has not decreased significantly.Bifidobacterium,Blautia only changed significantly in the early stages of the disease.Enterococcus are also significantly higher in patients with a long course of disease than short-term diseases and healthy controls,Part 21.The expression of CD 11 c+CD 11 b+DCs and CD 11 c+103+DCs in the colon of DSS-induced colitis mice was significantly lower than that of normal control mice(P<0.05)In addition,the intervention of L.reuteri promoted the proportion of CD11c+CD11b+DCs and CD11c+103+DCs.These results indicate that L.reuteri can activate the protective effects of tolerant DCs,and reduce DSS-induced intestinal inflammatory immune responses2.TNBS low-grade inflammatory pathology has no obvious changes,but inflammatory cytokines still increase.C.butyricum and butyrate can reduce the expression of IL-17A,and also significantly increased the spleen CD11c+CD11b+DCs IL-10 secretion.SignificanceThis study combine changes in the intestinal flora of UC with the duration of the disease,which may help guide the staging of UC.Explore the effects of probiotic interventions on the intestinal innate immune status in different disease periods.The disadvantage of this subject is that the specific action of probiotics on DCs and the impact on the intestinal flora after intervention still need to be further explored.更多还原