本文選題：類風濕關節(jié)炎 + 膠原誘導性關節(jié)炎　； 參考：《第三軍醫(yī)大學》2017年博士論文

【摘要】：背景:類風濕關節(jié)炎(rheumatoid arthritis,RA)是一種慢性、全身性的自身免疫性疾病,以關節(jié)疼痛、骨和軟骨的侵蝕為特征,最終可發(fā)展為功能性殘疾,嚴重威脅人類健康。然而RA發(fā)病機制并不清楚,遺傳因素并不能完全解釋其發(fā)病原因。多項研究表明RA患者的腸道菌群的不同于健康人:學者認為腸道菌群及其代謝產物或許能夠參與自身免疫性疾病的發(fā)病[1],腸道細菌可能通過分子模擬機制參與RA發(fā)病[2]。細菌可以誘發(fā)無菌小鼠的關節(jié)炎癥狀[3,4],然而另有研究發(fā)現(xiàn)補充益生菌可減輕關節(jié)炎癥狀和改善炎癥狀態(tài)[5,6],由此可見腸道細菌對RA疾病的影響尚存有爭議。我們前期研究證明RA的腸道菌群發(fā)生改變,然而在RA發(fā)病前后患者腸道菌群如何變化,及此種變化如何影響RA病情均不清楚。由于宿主的遺傳背景和棲息地環(huán)境均能影響腸道菌群[7],并且收集RA患者發(fā)病前的腸道菌群具有一定難度,使研究腸道菌群在RA發(fā)病中的作用較為困難。然而運用無菌動物和自身免疫性關節(jié)炎模型可以解決上述困擾,有利于研究腸道菌群在關節(jié)炎發(fā)病中的作用。膠原誘導性關節(jié)炎(collagen-induced arthritis,CIA)模型是一種自身免疫性關節(jié)炎模型,DBA/1小鼠的CIA發(fā)病率約為80%[8],這種小鼠模型與RA的免疫學和病理學特征相似,因此它已被廣泛使用,并作為研究RA發(fā)病機制和測試新療法的重要工具。目的:我們利用II型膠原免疫DBA/1小鼠,比較CIA易感小鼠(即患關節(jié)炎組)與CIA抵抗小鼠(即未患關節(jié)炎組)的腸道菌群差異,比較CIA易感小鼠關節(jié)炎發(fā)病前后其腸道菌群變化。尋找在CIA發(fā)病中起關鍵作用的細菌,然后定植腸道菌群至無菌小鼠,使用膠原免疫小鼠,調查腸道菌群對膠原誘導性關節(jié)炎發(fā)病的影響。明確腸道菌群的致病或保護作用,為深入了解RA的發(fā)病機制及治療RA提供新思路。我們前期研究還發(fā)現(xiàn)唾液乳桿菌(Lactobacillus salivarius)在RA患者腸道中檢出率高于健康人。唾液乳桿菌對RA發(fā)病起保護作用還是致病作用并不清楚,值得進一步研究。我們分離RA患者的唾液乳桿菌研究其對CIA小鼠發(fā)病的影響。第一部分分析膠原誘導性關節(jié)炎小鼠的腸道菌群特征方法:使用II型膠原免疫DBA/1小鼠,構建CIA模型,運用16S r RNA測序技術分析CIA易感小鼠、CIA抵抗小鼠和未免疫小鼠的腸道菌群特征。首先分析各組腸道菌群的α多樣性差異,包括小鼠腸道菌群的Reads數(shù)、操作分類單元數(shù)、豐富度指數(shù)(ACE和Chao)和多樣性指數(shù)(Shannon和Simpson)的差異;其次分析各組小鼠腸道菌群的β多樣性差異;最后分析各組小鼠腸道菌群種類、數(shù)量的差異。結果:α多樣性分析:在使用II型膠原免疫DBA/1小鼠后,無論關節(jié)炎發(fā)病與否,小鼠腸道菌群序列數(shù)目均顯著減少。在關節(jié)炎發(fā)病后,CIA易感小鼠腸道菌群的豐富度指數(shù)低于CIA抵抗小鼠和正常對照組。然而伴隨著關節(jié)炎發(fā)病,CIA易感小鼠的腸道菌群多樣性指數(shù)較發(fā)病前顯著增加。β多樣性分析:相似性分析表明在關節(jié)炎發(fā)病前,CIA易感小鼠和抵抗小鼠的腸道菌群結構存在差異(R=0.375,P0.01)。腸道菌群數(shù)量差異:在關節(jié)炎發(fā)病前CIA易感小鼠腸道中脫硫弧菌科(Desulfovibrionaceae)和毛螺旋菌科(Lachnospiraceae)細菌的含量顯著少于CIA抵抗小鼠,而乳桿菌科(Lactobacillaceae)細菌在CIA易感小鼠腸道中的含量比抵抗小鼠更加豐富。伴隨關節(jié)炎發(fā)病,CIA易感小鼠腸道中類桿菌科(Bacteroidaceae),毛螺旋菌科(Lachnospiraceae)和S24-7科細菌含量較發(fā)病前顯著增加。結論:在膠原免疫后DBA/1小鼠的腸道菌群發(fā)生改變;可分為CIA易感菌群和CIA抵抗菌群;CIA易感菌群的多樣性及菌群結構不同于CIA抵抗菌群;伴隨關節(jié)炎發(fā)生小鼠腸道菌群發(fā)生改變。第二部分腸道菌群影響無菌小鼠膠原誘導性關節(jié)炎發(fā)病的作用及機制方法:分別定植CIA易感小鼠和CIA抵抗小鼠的腸道菌群至無菌小鼠,然后使用膠原免疫小鼠,比較關節(jié)炎發(fā)病率及嚴重程度,檢測外周血中TNF-a、IL-10和IL-17水平,分析脾臟CD3-CD11c+淋巴細胞、CD3-CD19+淋巴細胞、CD4+T細胞、CD8+T細胞、Th1、Th17和Treg的數(shù)量。結果:16S r RNA測序發(fā)現(xiàn)受體的腸道菌群特征符合供體的腸道菌群特征,表明腸道菌群成功定植。CIA易感菌群較CIA抵抗菌群更能增加關節(jié)炎的發(fā)病率和嚴重程度。定植CIA易感菌群組小鼠外周血中IL-17濃度顯著高于對照組。定植CIA易感腸道菌群組小鼠脾臟CD8+T細胞比例增高、而CD3-CD11c+淋巴細胞和CD3-CD19+淋巴細胞比例顯著低于定植CIA抵抗菌群組。定植CIA易感菌群組小鼠的脾臟Th17細胞比例高于定植CIA抵抗菌群組,而Treg細胞比例顯著低于定植CIA抵抗菌群組。結論:腸道菌群影響CIA的易感性和抵抗性,腸道菌群可能通過影響Th17/Treg參與CIA發(fā)病。第三部分類風濕關節(jié)炎患者腸道乳桿菌對膠原誘導性關節(jié)炎的影響及機制研究方法:從RA患者腸道中分離培養(yǎng)唾液乳桿菌(L.salivarius UCC118)和植物乳桿菌(L.plantarum WCFS1)。分別定植上述兩種細菌至DBA/1小鼠,從初次免疫前2周開始灌胃,持續(xù)5周。記錄關節(jié)炎評分,觀察組織病理學改變,通過Micro-CT評價骨侵蝕,檢測外周血中細胞因子TNF-a、IL-10和IL-17水平,分析脾臟中Th17細胞和Treg細胞的數(shù)量。結果:與灌胃磷酸鹽緩沖液(Phosphate Buffered Saline,PBS)組相比,灌胃唾液乳桿菌或植物乳桿菌后CIA小鼠有較低的關節(jié)炎評分,表現(xiàn)出較弱的滑膜炎癥及較少的骨侵蝕。灌胃唾液乳桿菌或植物乳桿菌能夠減少脾臟Th17而增加脾臟Treg比例。灌胃唾液乳桿菌后CIA小鼠外周血中細胞因子IL-10的水平顯著增高。結論:從RA患者腸道分離的唾液乳桿菌和植物乳桿菌對CIA小鼠的慢性炎癥具有保護作用。唾液乳桿菌更能顯著緩解CIA小鼠關節(jié)炎病情,為臨床上使用益生菌制劑改善RA癥狀提供新思路。
[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.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R593.22

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