肠道菌群在胶原诱导性关节炎中的作用及机制研究

发布时间：2018-04-25 00:08

本文选题：类风湿关节炎 + 胶原诱导性关节炎　；参考：《第三军医大学》2017年博士论文

【摘要】：背景:类风湿关节炎(rheumatoid arthritis,RA)是一种慢性、全身性的自身免疫性疾病,以关节疼痛、骨和软骨的侵蚀为特征,最终可发展为功能性残疾,严重威胁人类健康。然而RA发病机制并不清楚,遗传因素并不能完全解释其发病原因。多项研究表明RA患者的肠道菌群的不同于健康人:学者认为肠道菌群及其代谢产物或许能够参与自身免疫性疾病的发病[1],肠道细菌可能通过分子模拟机制参与RA发病[2]。细菌可以诱发无菌小鼠的关节炎症状[3,4],然而另有研究发现补充益生菌可减轻关节炎症状和改善炎症状态[5,6],由此可见肠道细菌对RA疾病的影响尚存有争议。我们前期研究证明RA的肠道菌群发生改变,然而在RA发病前后患者肠道菌群如何变化,及此种变化如何影响RA病情均不清楚。由于宿主的遗传背景和栖息地环境均能影响肠道菌群[7],并且收集RA患者发病前的肠道菌群具有一定难度,使研究肠道菌群在RA发病中的作用较为困难。然而运用无菌动物和自身免疫性关节炎模型可以解决上述困扰,有利于研究肠道菌群在关节炎发病中的作用。胶原诱导性关节炎(collagen-induced arthritis,CIA)模型是一种自身免疫性关节炎模型,DBA/1小鼠的CIA发病率约为80%[8],这种小鼠模型与RA的免疫学和病理学特征相似,因此它已被广泛使用,并作为研究RA发病机制和测试新疗法的重要工具。目的:我们利用II型胶原免疫DBA/1小鼠,比较CIA易感小鼠(即患关节炎组)与CIA抵抗小鼠(即未患关节炎组)的肠道菌群差异,比较CIA易感小鼠关节炎发病前后其肠道菌群变化。寻找在CIA发病中起关键作用的细菌,然后定植肠道菌群至无菌小鼠,使用胶原免疫小鼠,调查肠道菌群对胶原诱导性关节炎发病的影响。明确肠道菌群的致病或保护作用,为深入了解RA的发病机制及治疗RA提供新思路。我们前期研究还发现唾液乳杆菌(Lactobacillus salivarius)在RA患者肠道中检出率高于健康人。唾液乳杆菌对RA发病起保护作用还是致病作用并不清楚,值得进一步研究。我们分离RA患者的唾液乳杆菌研究其对CIA小鼠发病的影响。第一部分分析胶原诱导性关节炎小鼠的肠道菌群特征方法:使用II型胶原免疫DBA/1小鼠,构建CIA模型,运用16S r RNA测序技术分析CIA易感小鼠、CIA抵抗小鼠和未免疫小鼠的肠道菌群特征。首先分析各组肠道菌群的α多样性差异,包括小鼠肠道菌群的Reads数、操作分类单元数、丰富度指数(ACE和Chao)和多样性指数(Shannon和Simpson)的差异;其次分析各组小鼠肠道菌群的β多样性差异;最后分析各组小鼠肠道菌群种类、数量的差异。结果:α多样性分析:在使用II型胶原免疫DBA/1小鼠后,无论关节炎发病与否,小鼠肠道菌群序列数目均显著减少。在关节炎发病后,CIA易感小鼠肠道菌群的丰富度指数低于CIA抵抗小鼠和正常对照组。然而伴随着关节炎发病,CIA易感小鼠的肠道菌群多样性指数较发病前显著增加。β多样性分析:相似性分析表明在关节炎发病前,CIA易感小鼠和抵抗小鼠的肠道菌群结构存在差异(R=0.375,P0.01)。肠道菌群数量差异:在关节炎发病前CIA易感小鼠肠道中脱硫弧菌科(Desulfovibrionaceae)和毛螺旋菌科(Lachnospiraceae)细菌的含量显著少于CIA抵抗小鼠,而乳杆菌科(Lactobacillaceae)细菌在CIA易感小鼠肠道中的含量比抵抗小鼠更加丰富。伴随关节炎发病,CIA易感小鼠肠道中类杆菌科(Bacteroidaceae),毛螺旋菌科(Lachnospiraceae)和S24-7科细菌含量较发病前显著增加。结论:在胶原免疫后DBA/1小鼠的肠道菌群发生改变;可分为CIA易感菌群和CIA抵抗菌群;CIA易感菌群的多样性及菌群结构不同于CIA抵抗菌群;伴随关节炎发生小鼠肠道菌群发生改变。第二部分肠道菌群影响无菌小鼠胶原诱导性关节炎发病的作用及机制方法:分别定植CIA易感小鼠和CIA抵抗小鼠的肠道菌群至无菌小鼠,然后使用胶原免疫小鼠,比较关节炎发病率及严重程度,检测外周血中TNF-a、IL-10和IL-17水平,分析脾脏CD3-CD11c+淋巴细胞、CD3-CD19+淋巴细胞、CD4+T细胞、CD8+T细胞、Th1、Th17和Treg的数量。结果:16S r RNA测序发现受体的肠道菌群特征符合供体的肠道菌群特征,表明肠道菌群成功定植。CIA易感菌群较CIA抵抗菌群更能增加关节炎的发病率和严重程度。定植CIA易感菌群组小鼠外周血中IL-17浓度显著高于对照组。定植CIA易感肠道菌群组小鼠脾脏CD8+T细胞比例增高、而CD3-CD11c+淋巴细胞和CD3-CD19+淋巴细胞比例显著低于定植CIA抵抗菌群组。定植CIA易感菌群组小鼠的脾脏Th17细胞比例高于定植CIA抵抗菌群组,而Treg细胞比例显著低于定植CIA抵抗菌群组。结论:肠道菌群影响CIA的易感性和抵抗性,肠道菌群可能通过影响Th17/Treg参与CIA发病。第三部分类风湿关节炎患者肠道乳杆菌对胶原诱导性关节炎的影响及机制研究方法:从RA患者肠道中分离培养唾液乳杆菌(L.salivarius UCC118)和植物乳杆菌(L.plantarum WCFS1)。分别定植上述两种细菌至DBA/1小鼠,从初次免疫前2周开始灌胃,持续5周。记录关节炎评分,观察组织病理学改变,通过Micro-CT评价骨侵蚀,检测外周血中细胞因子TNF-a、IL-10和IL-17水平,分析脾脏中Th17细胞和Treg细胞的数量。结果:与灌胃磷酸盐缓冲液(Phosphate Buffered Saline,PBS)组相比,灌胃唾液乳杆菌或植物乳杆菌后CIA小鼠有较低的关节炎评分,表现出较弱的滑膜炎症及较少的骨侵蚀。灌胃唾液乳杆菌或植物乳杆菌能够减少脾脏Th17而增加脾脏Treg比例。灌胃唾液乳杆菌后CIA小鼠外周血中细胞因子IL-10的水平显著增高。结论:从RA患者肠道分离的唾液乳杆菌和植物乳杆菌对CIA小鼠的慢性炎症具有保护作用。唾液乳杆菌更能显著缓解CIA小鼠关节炎病情,为临床上使用益生菌制剂改善RA症状提供新思路。
[Abstract]:Background: rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by joint pain, bone and cartilage erosion, which eventually develops into functional disability and is a serious threat to human health. However, the pathogenesis of RA is not clear and genetic factors can not fully explain the cause of the disease. A number of studies are not fully explained. The study shows that the intestinal flora of RA patients is different from that of healthy people: scholars believe that intestinal flora and its metabolites may be able to participate in the pathogenesis of autoimmune disease [1]. Intestinal bacteria may participate in the pathogenesis of RA by molecular simulation mechanism to induce the arthritis symptom [3,4] in aseptic mice. However, other studies have found complementary probiotics. The bacteria can reduce the symptoms of arthritis and improve the inflammatory state of [5,6]. This shows that the effect of intestinal bacteria on RA disease is still controversial. Our previous studies have proved that the intestinal flora of RA changes, however, how the intestinal flora changes before and after the onset of RA, and how this change affects the RA condition is not clear. The genetic background of the host is the genetic background of the host. And the habitat environment can affect the intestinal flora [7], and it is difficult to collect intestinal microflora before the onset of RA, which makes it difficult to study the intestinal flora in the pathogenesis of RA. However, the use of aseptic animals and autoimmune arthritis model can solve these problems, which is beneficial to the study of intestinal flora in the pathogenesis of arthritis. Collagen-induced arthritis (CIA) model is an autoimmune arthritis model, and the incidence of CIA in DBA/1 mice is about 80%[8]. This mouse model is similar to the immunological and pathological features of RA, so it has been widely used as an important tool to study the pathogenesis of RA and to test the new therapy. Objective: we use type II collagen to immunize DBA/1 mice to compare the intestinal microflora of CIA susceptible mice (that of arthritis group) and CIA resistant mice (that is, no arthritis group), to compare the intestinal microflora changes in CIA susceptible mice before and after the onset of arthritis. To search for the bacteria that play a key role in the pathogenesis of CIA, and then to colonize the intestinal flora to aseptic small bacteria. Mice were immunized with collagen to investigate the effect of intestinal flora on the pathogenesis of collagen induced arthritis. To clarify the pathogenic or protective effects of intestinal flora in order to understand the pathogenesis of RA and to provide new ideas for the treatment of RA. Our previous study also found that the detection rate of Lactobacillus salivarius in the intestinal tract of RA patients was higher than that of healthy. The protective effect of Lactobacillus saliva on the pathogenesis of RA is not clear, it is worth further study. We isolate the effect of Lactobacillus saliva from RA patients on the pathogenesis of CIA mice. The first part analyses the intestinal microflora characteristics of collagen induced arthritis mice: using II collagen to immunization DBA/1 mice and construct CIA model. 16S R RNA sequencing technology was used to analyze the intestinal microflora characteristics of CIA susceptible mice, CIA resistant mice and unimmunized mice. Firstly, the differences in the alpha diversity of intestinal microflora were analyzed, including the Reads number of intestinal flora, the number of operation classification units, the diversity of the richness index (ACE and Chao) and the diversity index (Shannon and Simpson); secondly, the difference in the diversity of the diversity index (Shannon and Simpson) of the intestinal microflora in mice was analyzed. The diversity of intestinal microflora in each group was analyzed. Finally, the diversity of intestinal microflora in each group was analyzed. Results: alpha diversity analysis: after the DBA/1 mice were immunized with II collagen, the number of intestinal microflora in mice decreased significantly. After the onset of arthritis, CIA was susceptible to the intestinal flora of mice. The richness index was lower than that of the CIA resistance mice and the normal control group. However, with the onset of arthritis, the intestinal microflora diversity index of CIA susceptible mice was significantly higher than that before the onset. The similarity analysis showed that before the onset of arthritis, the intestinal flora structure of CIA susceptible mice and resistance mice was different (R=0.375, P0.01). Differences in the number of intestinal microflora: before the onset of arthritis, the content of Desulfovibrionaceae and Lachnospiraceae in the intestinal tract of CIA susceptible mice was significantly less than that of CIA resistance mice, and the content of Lactobacillaceae bacteria in the intestinal tract of CIA susceptible mice was more abundant than that of the resistance mice. Inflammation, CIA susceptible mice intestinal bacilli family (Bacteroidaceae), Lachnospiraceae and S24-7 bacteria content before the onset of significantly increased. Conclusion: after the collagen immunization of DBA/1 mice intestinal flora change, can be divided into CIA susceptible bacteria group and CIA resistance bacteria group; CIA susceptible bacteria diversity and the structure of different bacteria groups The intestinal flora of mice with CIA resistance was changed. The effect and mechanism of second parts of intestinal flora on the pathogenesis of collagen induced arthritis in aseptic mice: colonization of intestinal flora of CIA susceptible mice and CIA resistant mice to aseptic mice, and then immunized with collagen in mice, the incidence of arthritis was compared. And the level of TNF-a, IL-10 and IL-17 in peripheral blood were detected. The number of spleen CD3-CD11c+ lymphocyte, CD3-CD19+ lymphocyte, CD4+T cell, CD8+T cell, Th1, Th17 and Treg were analyzed. The group of CIA resistant bacteria group increased the incidence and severity of arthritis more. The concentration of IL-17 in the peripheral blood of the colonized CIA susceptible group mice was significantly higher than that of the control group. The proportion of CD8+T cells in the spleen of the colonized CIA susceptible group mice was higher, while the proportion of CD3-CD11c+ and CD3-CD19+ lymphoblastic cells was significantly lower than that of the colonized CIA resistance group. The proportion of Th17 cells in the spleen of CIA susceptible group was higher than that of the colonization of CIA resistant bacteria group, and the proportion of Treg cells was significantly lower than that of the colonization of CIA resistance bacteria group. Conclusion: intestinal flora affects the susceptibility and resistance of CIA, and intestinal flora may be involved in CIA disease by influencing Th17/Treg. The third part of rheumatoid arthritis patients enteric milk The effect and mechanism of bacilli on collagen induced arthritis: isolation and culture of Lactobacillus sialus (L.salivarius UCC118) and Lactobacillus plantarum (L.plantarum WCFS1) from the intestinal tract of RA patients. The above two kinds of bacteria were planted to DBA/1 mice, respectively, from the first 2 weeks before the first immunization, for 5 weeks. The arthritis score was recorded and the tissue disease was observed. By evaluating bone erosion by Micro-CT, the levels of cytokines TNF-a, IL-10 and IL-17 in peripheral blood were measured, and the number of Th17 cells and Treg cells in the spleen was analyzed. Results: compared with the gastric phosphate buffer solution (Phosphate Buffered Saline, PBS), there was a lower osteoarthritis evaluation in the group of Lactobacillus saliva or Lactobacillus plantarum. It showed weak synovitis and less bone erosion. Lactobacillus saliva or Lactobacillus plantarum could reduce the spleen Th17 and increase the proportion of spleen Treg. The level of cytokine IL-10 in the peripheral blood of CIA mice increased significantly after gavage of Lactobacillus saliva from Lactobacillus saliva from CIA mice. Conclusion: Lactobacillus and Lactobacillus plantarum isolated from the intestinal tract of RA patients were CIA Chronic inflammation in mice has a protective effect. Lactobacillus sialiti can significantly alleviate the arthritis in CIA mice, and provide a new idea for the clinical use of probiotics to improve the symptoms of RA.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R593.22

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