技术领域technical field
本发明涉及微生物学领域，具体地，涉及多氏拟杆菌在治疗或预防类风湿性关节炎或其相关疾病中的用途，也涉及包含多氏拟杆菌的药物组合物、药物、食品和饲料。The present invention relates to the field of microbiology, in particular, relates to the use of Bacteroides multani in the treatment or prevention of rheumatoid arthritis or related diseases, and also relates to pharmaceutical compositions, medicines, foods and feeds containing Bacteroides multani.
背景技术Background technique
类风湿性关节炎(rheumatoid arthritis，RA)简称类风湿，是一种全身性自身免疫性疾病，主要累及关节滑膜，其次为浆膜、心、肺、血管、神经等结缔组织。急性期炎症主要表现为滑膜血管水肿、滑膜炎，若未被及时诊断和治疗，急性炎症反应消退，渗出逐渐吸收，滑膜中血管翳形成，滑膜细胞显著增生。继而引起关节软骨破坏，骨组织炎症、坏死和纤维结缔组织增生。而后发生钙化出现纤维性和骨性关节硬化，关节腔显著狭窄或完全消失。此期慢性炎症过程造成的关节功能障碍，在很大程度上难以恢复，可致永久性残疾。全世界每100～150人中就有1人患有RA，我国患病率为0.32％～0.36％。RA在各年龄皆可发病，成人中多发于中年女性，男女之比约1:3。2003年据WHO报道，在良好的医疗条件下，每人每年的治疗费用约为6000美元，由该病造成的间接损失也不容忽视。Rheumatoid arthritis (RA), referred to as rheumatoid, is a systemic autoimmune disease that mainly involves joint synovium, followed by connective tissues such as serosa, heart, lung, blood vessels, and nerves. In the acute stage, inflammation mainly manifests as synovial angioedema and synovitis. If not diagnosed and treated in time, the acute inflammatory response subsides, the exudate is gradually absorbed, pannus forms in the synovium, and synovial cells proliferate significantly. Then it causes the destruction of articular cartilage, bone tissue inflammation, necrosis and hyperplasia of fibrous connective tissue. Then calcification occurs, fibrous and bony joint sclerosis occurs, and the joint cavity is significantly narrowed or completely disappeared. The joint dysfunction caused by the chronic inflammatory process in this stage is difficult to recover to a large extent and can cause permanent disability. One person in every 100-150 people in the world suffers from RA, and the prevalence rate in my country is 0.32%-0.36%. RA can occur at all ages, and it mostly occurs in middle-aged women in adults, with a male to female ratio of about 1:3. According to WHO reports in 2003, under good medical conditions, the annual treatment cost per person is about 6,000 US dollars. The indirect losses caused by the disease cannot be ignored.
目前，西医主要采用药物治疗和基因治疗等方法。常用西药主要有非甾体抗炎药(NSAIDs)、糖皮质激素、慢作用药、免疫抑制剂及生物制剂等。但西药普遍具有价格高、毒副作用较大的缺点，所以限制了它们的应用。基因治疗的选择性高，其可针对疾病从分子水平进行治疗，同时可以通过限制基因的长期高水平局部表达，为RA患者的治疗开辟了新的途径。但是，目前基因治疗还处于起步阶段，仍存在较多的问题。如缺乏理想的载体、不同的宿主对载体产生不同的免疫反应、导入的基因在机体内是否能稳定表达等都还有待进一步研究探讨。此外，基因治疗的安全性、治疗费用及伦理学问题等也是此项研究需要解决的问题。At present, western medicine mainly adopts methods such as drug therapy and gene therapy. Commonly used western medicines mainly include non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, slow-acting drugs, immunosuppressants and biological agents. However, western medicine generally has the disadvantages of high price and high toxicity and side effects, so their application is limited. The selectivity of gene therapy is high, it can treat the disease from the molecular level, and at the same time, it can open up a new way for the treatment of RA patients by limiting the long-term high-level local expression of genes. However, gene therapy is still in its infancy, and there are still many problems. For example, the lack of ideal vectors, different hosts have different immune responses to vectors, and whether the introduced genes can be stably expressed in the body, etc., still need further research and exploration. In addition, the safety, treatment cost and ethical issues of gene therapy are also issues that need to be resolved in this study.
已有证据表明，肠道微生物与系统免疫应答和RA存在一定的联系。乳酸菌(lacticacid bacteria，LAB)可以在不影响调控细胞活素水平的前提下降低促炎细胞活素水平，从而发挥抗炎作用，减轻RA症状。因此可以推测，一些使肠道微生态恢复常态的治疗手段，在恢复肠道及免疫系统功能的同时，可能也会对减轻RA症状发挥一定的作用。研究证实，一种LAB(Bacillus coagulans GBI-30,6086)可能成为一种安全高效的减轻RA症状的辅助药物There is already evidence that gut microbes are linked to systemic immune responses and RA. Lactic acid bacteria (LAB) can reduce the level of pro-inflammatory cytokines without affecting the regulation of cytokine levels, thereby exerting anti-inflammatory effects and reducing RA symptoms. Therefore, it can be speculated that some treatment methods to restore the intestinal microecology to normal may play a certain role in alleviating the symptoms of RA while restoring the intestinal and immune system functions. The study confirmed that a LAB (Bacillus coagulans GBI-30, 6086) may become a safe and effective adjuvant drug for relieving RA symptoms
由于病因与发病机制尚未完全明了，RA的治疗虽取得一定进展，但至今仍处于控制或缓解症状阶段，并无有效治疗或治愈方法。因此本领域迫切需要开发一种有效的新的，无毒副作用的，用于治疗和预防类风湿性关节炎及其相关疾病的药物。Since the etiology and pathogenesis are not yet fully understood, although some progress has been made in the treatment of RA, it is still in the stage of controlling or relieving symptoms, and there is no effective treatment or cure. Therefore, there is an urgent need in this field to develop a new effective drug without toxic and side effects for the treatment and prevention of rheumatoid arthritis and related diseases.
发明内容Contents of the invention
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的一个目的在于提出多氏拟杆菌在治疗或预防类风湿性关节炎或其相关疾病中的用途。The present invention aims to solve one of the technical problems in the related art at least to a certain extent. Therefore, one object of the present invention is to propose the use of Bacteroides multineri in the treatment or prevention of rheumatoid arthritis or related diseases.
在本发明的第一方面，本发明提供了多氏拟杆菌在制备药物中的用途，所述药物用于治疗或预防类风湿性关节炎或其相关疾病。发明人发现，通过给动物提供该药物，能够有效发挥抗炎的功效，关节发红和肿胀的症状得到有效改善，关节炎症状明显减轻，即能够有效治疗或预防类风湿性关节炎或其相关疾病。In the first aspect of the present invention, the present invention provides the use of Bacteroides tenerii in the preparation of medicaments for treating or preventing rheumatoid arthritis or related diseases. The inventors found that by providing the drug to animals, the anti-inflammatory effect can be effectively exerted, the symptoms of joint redness and swelling can be effectively improved, and the symptoms of arthritis can be significantly reduced, that is, rheumatoid arthritis or its related diseases can be effectively treated or prevented. disease.
根据本发明的实施例，所述多氏拟杆菌为Bacteroides dorei。由此，能够有效发挥治疗或预防类风湿性关节炎或其相关疾病的功效。According to an embodiment of the present invention, the Bacteroides dorei is Bacteroides dorei. Thus, the effect of treating or preventing rheumatoid arthritis or related diseases can be effectively exerted.
根据本发明的实施例，所述多氏拟杆菌为选自Bacteroides dorei DSM 17855、Bacteroides dorei JCM 13472和Bacteroides dorei BD1中的至少一种。由此，治疗或预防类风湿性关节炎或其相关疾病的效果较佳。According to an embodiment of the present invention, the Bacteroides dorei is at least one selected from Bacteroides dorei DSM 17855, Bacteroides dorei JCM 13472 and Bacteroides dorei BD1. Therefore, the effect of treating or preventing rheumatoid arthritis or related diseases is better.
在本发明的第二方面，本发明提供了一种药物组合物。根据本发明的实施例，该药物组合物包括：多氏拟杆菌；以及药学上可接受的辅料。发明人惊喜地发现，通过对动物提供该药物组合物，能够明显抑制关节炎的发生，关节发红和肿胀明显减退，关节炎症状明显减轻，动物行动灵活，表明该药物组合物能够有效治疗或预防类风湿性关节炎或其相关疾病。In a second aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes: Bacteroides multuri; and pharmaceutically acceptable auxiliary materials. The inventors are surprised to find that by providing the pharmaceutical composition to animals, the occurrence of arthritis can be significantly inhibited, the redness and swelling of the joints can be significantly reduced, the symptoms of arthritis can be significantly reduced, and the animals can move flexibly, indicating that the pharmaceutical composition can effectively treat or Prevention of rheumatoid arthritis or related diseases.
根据本发明的实施例，所述多氏拟杆菌为Bacteroides dorei。由此，能够有效发挥治疗或预防类风湿性关节炎或其相关疾病的功效。According to an embodiment of the present invention, the Bacteroides dorei is Bacteroides dorei. Thus, the effect of treating or preventing rheumatoid arthritis or related diseases can be effectively exerted.
根据本发明的实施例，所述多氏拟杆菌为选自Bacteroides dorei DSM 17855、Bacteroides dorei JCM 13472和Bacteroides dorei BD1中的至少一种。由此，治疗或预防类风湿性关节炎或其相关疾病的效果较佳。According to an embodiment of the present invention, the Bacteroides dorei is at least one selected from Bacteroides dorei DSM 17855, Bacteroides dorei JCM 13472 and Bacteroides dorei BD1. Therefore, the effect of treating or preventing rheumatoid arthritis or related diseases is better.
根据本发明的实施例，当所述药物组合物呈固态时，所述药物组合物包含1×10-1×1020cfu/g的所述多氏拟杆菌，当所述药物组合物呈液态时，所述药物组合物包含1×10-1×1020cfu/mL的所述多氏拟杆菌。由此，抑制关节炎发生和改善关节炎症状的效果明显，如果多氏拟杆菌含量过低，治疗或预防类风湿性关节炎的效果不理想，如果多氏拟杆菌含量过高，则治疗或预防类风湿性关节炎的的效果无明显提高，造成浪费。According to an embodiment of the present invention, when the pharmaceutical composition is in a solid state, the pharmaceutical composition contains 1×10-1×1020 cfu/g of the Bacteroides multurii, and when the pharmaceutical composition is in a liquid state When, the pharmaceutical composition comprises 1×10-1×1020 cfu/mL of the Bacteroides multani. Thus, the effect of inhibiting the occurrence of arthritis and improving the symptoms of arthritis is obvious. If the content of Bacteroides dorsii is too low, the effect of treating or preventing rheumatoid arthritis is not satisfactory. The effect of preventing rheumatoid arthritis is not significantly improved, resulting in waste.
根据本发明的一个优选实施例，当所述药物组合物呈固态时，所述药物组合物包含1×104-1×1015cfu/g的所述多氏拟杆菌，当所述药物组合物呈液态时，所述药物组合物包含1×104-1×1015cfu/mL的所述多氏拟杆菌。According to a preferred embodiment of the present invention, when the pharmaceutical composition is in a solid state, the pharmaceutical composition contains 1×104 -1×1015 cfu/g of the Bacteroides multellii, when the pharmaceutical composition When the substance is in a liquid state, the pharmaceutical composition comprises 1×104 -1×1015 cfu/mL of the Bacteroides multani.
根据本发明的另一个优选实施例，当所述药物组合物呈固态时，所述药物组合物包含1×106-1×1011cfu/g，的所述多氏拟杆菌，当所述药物组合物呈液态时，所述药物组合物包含1×106-1×1011cfu/mL的所述多氏拟杆菌。According to another preferred embodiment of the present invention, when the pharmaceutical composition is in a solid state, the pharmaceutical composition contains 1×106 -1×1011 cfu/g of the Bacteroides multurii, when the When the pharmaceutical composition is in a liquid state, the pharmaceutical composition contains 1×106 -1×1011 cfu/mL of the Bacteroides multani.
根据本发明的实施例，所述药学上可接受的辅料为选自载体、赋形剂、稀释剂、润滑剂、润湿剂、乳化剂、悬浮液稳定剂、防腐剂、甜味剂以及香料中的至少一种。According to an embodiment of the present invention, the pharmaceutically acceptable adjuvant is selected from carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and spices at least one of the
根据本发明的实施例，所述药学上可接受的辅料为选自乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖、淀粉、阿拉伯胶、磷酸钙、藻酸盐、明胶、硅酸钙、细结晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁和矿物油中的至少一种。According to an embodiment of the present invention, the pharmaceutically acceptable auxiliary material is selected from lactose, glucose, sucrose, sorbitol, mannose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, fine At least one of crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.
在本发明的第三方面，本发明提供了一种药物。根据本发明的实施例，该药物包括：前面所述的药物组合物。通过对动物提供该药物，能够明显抑制关节炎的发生，关节发红和肿胀明显减退，关节炎症状明显减轻，动物行动灵活，表明该药物能够有效治疗或预防类风湿性关节炎或其相关疾病。In a third aspect of the invention, the invention provides a medicament. According to an embodiment of the present invention, the medicine includes: the aforementioned pharmaceutical composition. By providing the drug to animals, the occurrence of arthritis can be significantly inhibited, the redness and swelling of the joints can be significantly reduced, the symptoms of arthritis can be significantly reduced, and the animals can move flexibly, indicating that the drug can effectively treat or prevent rheumatoid arthritis or related diseases .
根据本发明的实施例，所述药物呈选自颗粒剂、胶囊、片剂、粉末剂、口服液、混悬液和乳剂中的至少一种制剂形式。由此，易于进行给药。According to an embodiment of the present invention, the medicine is in the form of at least one preparation selected from granules, capsules, tablets, powders, oral liquids, suspensions and emulsions. Thus, administration is facilitated.
在本发明的第四方面，本发明提供了一种食品。根据本发明的实施例，该食品包括：多氏拟杆菌；以及食品学上可接受的辅料。发明人发现，通过对动物提供该食品，能够明显抑制关节炎的发生，关节发红和肿胀明显减退，关节炎症状明显减轻，动物行动灵活，表明该食品能够有效治疗或预防类风湿性关节炎或其相关疾病。In a fourth aspect of the invention, the invention provides a food product. According to an embodiment of the present invention, the food includes: Bacteroides multuri; and food acceptable auxiliary materials. The inventor found that by providing the food to animals, the occurrence of arthritis can be significantly inhibited, the redness and swelling of the joints can be significantly reduced, the symptoms of arthritis can be significantly reduced, and the animals can move flexibly, indicating that the food can effectively treat or prevent rheumatoid arthritis or its related diseases.
需要说明的是，本发明所使用的术语“食品”的种类不受特别限制，可以为任何已知的食品，包括但不限于奶制品、饼干、糕点、饮料、保健品等。It should be noted that the type of the term "food" used in the present invention is not particularly limited, and may be any known food, including but not limited to dairy products, biscuits, cakes, beverages, health products, etc.
根据本发明的实施例，所述多氏拟杆菌为Bacteroides dorei。由此，能够有效发挥治疗或预防类风湿性关节炎或其相关疾病的功效。According to an embodiment of the present invention, the Bacteroides dorei is Bacteroides dorei. Thus, the effect of treating or preventing rheumatoid arthritis or related diseases can be effectively exerted.
根据本发明的实施例，所述多氏拟杆菌为选自Bacteroides dorei DSM 17855、Bacteroides dorei JCM 13472和Bacteroides dorei BD1中的至少一种。由此，治疗或预防类风湿性关节炎或其相关疾病的效果较佳。According to an embodiment of the present invention, the Bacteroides dorei is at least one selected from Bacteroides dorei DSM 17855, Bacteroides dorei JCM 13472 and Bacteroides dorei BD1. Therefore, the effect of treating or preventing rheumatoid arthritis or related diseases is better.
根据本发明的实施例，当所述食品呈固态时，所述食品包含1×10-1×1020cfu/g的所述多氏拟杆菌，当所述食品呈液态时，所述食品包含1×10-1×1020cfu/mL的所述多氏拟杆菌。由此，抑制关节炎发生和改善关节炎症状的效果明显，如果多氏拟杆菌含量过低，治疗或预防类风湿性关节炎的效果不理想，如果多氏拟杆菌含量过高，则治疗或预防类风湿性关节炎的的效果无明显提高，造成浪费。According to an embodiment of the present invention, when the food is in a solid state, the food contains 1×10-1×1020 cfu/g of the Bacteroides multellii, and when the food is in a liquid state, the food contains 1×10-1×1020 cfu/mL of the Bacteroides multurii. Thus, the effect of inhibiting the occurrence of arthritis and improving the symptoms of arthritis is obvious. If the content of Bacteroides dorsii is too low, the effect of treating or preventing rheumatoid arthritis is not satisfactory. The effect of preventing rheumatoid arthritis is not significantly improved, resulting in waste.
根据本发明的一个优选实施例，当所述食品呈固态时，所述食品包含1×104-1×1015cfu/g的所述多氏拟杆菌，当所述食品呈液态时，所述食品包含1×104-1×1015cfu/mL的所述多氏拟杆菌。According to a preferred embodiment of the present invention, when the food is in a solid state, the food contains 1×104 -1×1015 cfu/g of the Bacteroides multurii; when the food is in a liquid state, the Said food contains 1×104 -1×1015 cfu/mL of said Bacteroides multani.
根据本发明的另一个优选实施例，当所述食品呈固态时，所述食品包含1×106-1×1011cfu/g，的所述多氏拟杆菌，当所述食品呈液态时，所述食品包含1×106-1×1011cfu/mL的所述多氏拟杆菌。According to another preferred embodiment of the present invention, when the food is in a solid state, the food contains 1×106 -1×1011 cfu/g, of the Bacteroides multurii, and when the food is in a liquid state , the food contains 1×106 -1×1011 cfu/mL of the Bacteroides multani.
根据本发明的实施例，所述食品学上可接受的辅料为选自载体、赋形剂、稀释剂、润滑剂、润湿剂、乳化剂、悬浮液稳定剂、防腐剂、甜味剂以及香料中的至少一种。According to an embodiment of the present invention, the food acceptable auxiliary material is selected from carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and at least one of the spices.
根据本发明的实施例，所述食品学上可接受的辅料为选自乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖、淀粉、阿拉伯胶、磷酸钙、藻酸盐、明胶、硅酸钙、细结晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁和矿物油中的至少一种。According to an embodiment of the present invention, the food acceptable auxiliary material is selected from lactose, glucose, sucrose, sorbitol, mannose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, At least one of fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate and mineral oil.
根据本发明的实施例，所述食品呈选自固体、乳品、溶液制品、粉末制品和悬浮液制品中的至少一种制剂形式。According to an embodiment of the present invention, the food is in the form of at least one formulation selected from solids, dairy products, solution products, powder products and suspension products.
在本发明的第五方面，本发明提供了一种饲料。根据本发明的实施例，该饲料包括：多氏拟杆菌。发明人发现，通过对动物提供该饲料，能够明显抑制关节炎的发生，动物关节发红和肿胀明显减退，关节炎症状明显减轻，动物行动灵活，表明该饲料能够有效治疗或预防类风湿性关节炎或其相关疾病。In a fifth aspect of the present invention, the present invention provides a feed. According to an embodiment of the present invention, the feed includes: Bacteroides multani. The inventor found that by providing the feed to animals, the occurrence of arthritis can be significantly inhibited, the redness and swelling of the animal joints are significantly reduced, the symptoms of arthritis are significantly reduced, and the animals are flexible, indicating that the feed can effectively treat or prevent rheumatoid arthritis. inflammation or related diseases.
根据本发明的实施例，所述多氏拟杆菌为Bacteroides dorei。由此，能够有效发挥治疗或预防类风湿性关节炎或其相关疾病的功效。According to an embodiment of the present invention, the Bacteroides dorei is Bacteroides dorei. Thus, the effect of treating or preventing rheumatoid arthritis or related diseases can be effectively exerted.
根据本发明的实施例，所述多氏拟杆菌为选自Bacteroides dorei DSM 17855、Bacteroides dorei JCM 13472和Bacteroides dorei BD1中的至少一种。由此，治疗或预防类风湿性关节炎或其相关疾病的效果较佳。According to an embodiment of the present invention, the Bacteroides dorei is at least one selected from Bacteroides dorei DSM 17855, Bacteroides dorei JCM 13472 and Bacteroides dorei BD1. Therefore, the effect of treating or preventing rheumatoid arthritis or related diseases is better.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.
附图说明Description of drawings
图1显示了根据本发明一个实施例，菌剂1、菌剂2和菌剂3对小鼠关节炎发病的影响宏观评分结果；Fig. 1 has shown according to one embodiment of the present invention, the impact macroscopic scoring result of bacterial agent 1, bacterial agent 2 and bacterial agent 3 on mouse arthritis pathogenesis;
图2显示了根据本发明一个实施例，菌剂1、菌剂2和菌剂3对关节炎小鼠脾细胞体外增殖的影响结果；以及Fig. 2 has shown according to one embodiment of the present invention, the effect of bacterial agent 1, bacterial agent 2 and bacterial agent 3 on the splenocyte proliferation of arthritis mice in vitro; and
图3显示了根据本发明一个实施例，菌剂1、菌剂2和菌剂3对关节炎小鼠细胞因子的影响结果。Fig. 3 shows the effect of bacterial agent 1, bacterial agent 2 and bacterial agent 3 on cytokines in arthritis mice according to an embodiment of the present invention.
具体实施方式Detailed ways
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York：Cold Spring HarborLaboratory Press，1989)中所述的条件，或按照《微生物：实验手册》(James Cappuccino和Natalie Sherman编，Pearson Education出版社)中所述的条件，或按照制造厂商所建议的条件。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to "Microbial: "Experimental Manual" (James Cappuccino and Natalie Sherman edited, Pearson Education Press), or in accordance with the conditions recommended by the manufacturer.
在本发明的第一方面，本发明提供了多氏拟杆菌在制备药物中的用途，所述药物用于治疗或预防类风湿性关节炎或其相关疾病。发明人发现，通过给动物提供该药物，能够有效发挥抗炎的功效，关节发红和肿胀的症状得到有效改善，关节炎症状明显减轻，即能够有效治疗或预防类风湿性关节炎或其相关疾病。In the first aspect of the present invention, the present invention provides the use of Bacteroides tenerii in the preparation of medicaments for treating or preventing rheumatoid arthritis or related diseases. The inventors found that by providing the drug to animals, the anti-inflammatory effect can be effectively exerted, the symptoms of joint redness and swelling can be effectively improved, and the symptoms of arthritis can be significantly reduced, that is, rheumatoid arthritis or its related diseases can be effectively treated or prevented. disease.
需要说明的是，本文中所使用的术语“动物”不受特别限制，可以为任何具有肠道的动物，优选哺乳动物，更优选人、兔类或鼠类。It should be noted that the term "animal" used herein is not particularly limited, and may be any animal with an intestinal tract, preferably a mammal, more preferably a human, a rabbit or a mouse.
根据本发明的实施例，所述多氏拟杆菌为Bacteroides dorei。由此，能够有效发挥治疗或预防类风湿性关节炎或其相关疾病的功效。According to an embodiment of the present invention, the Bacteroides dorei is Bacteroides dorei. Thus, the effect of treating or preventing rheumatoid arthritis or related diseases can be effectively exerted.
根据本发明的实施例，所述多氏拟杆菌为选自Bacteroides dorei DSM 17855、Bacteroides dorei JCM 13472和Bacteroides dorei BD1中的至少一种。由此，治疗或预防类风湿性关节炎或其相关疾病的效果较佳。According to an embodiment of the present invention, the Bacteroides dorei is at least one selected from Bacteroides dorei DSM 17855, Bacteroides dorei JCM 13472 and Bacteroides dorei BD1. Therefore, the effect of treating or preventing rheumatoid arthritis or related diseases is better.
根据本发明的实施例，受试动物经过造模形成关节炎模型动物，模型动物摄入Bacteroides dorei DSM 17855、Bacteroides dorei JCM 13472和Bacteroides dorei BD1，均能够明显改善动物关节发红和肿胀状况，减轻关节炎症状。根据本发明的实施例，分别经菌株Bacteroides dorei DSM 17855、Bacteroides dorei JCM 13472和Bacteroidesdorei BD1来治疗的关节炎DBA/1J小鼠，与未接受治疗的对照组相比，关节发红和肿胀明显消退，关节炎症状明显减轻。由此，表明所述菌株能够用以预防和治疗类风湿性关节炎或其相关疾病。According to the embodiments of the present invention, the test animals were modeled to form arthritis model animals, and the model animals ingested Bacteroides dorei DSM 17855, Bacteroides dorei JCM 13472 and Bacteroides dorei BD1, all of which could significantly improve the redness and swelling of the animals' joints, relieve Arthritis symptoms. According to an embodiment of the present invention, the arthritic DBA/1J mice treated with the strains Bacteroides dorei DSM 17855, Bacteroides dorei JCM 13472 and Bacteroides dorei BD1, respectively, compared with the untreated control group, the joint redness and swelling significantly subsided , Arthritis symptoms significantly reduced. Thus, it is shown that the strain can be used to prevent and treat rheumatoid arthritis or related diseases.
在本发明的第二方面，本发明提供了一种药物组合物。根据本发明的实施例，该药物组合物包括：多氏拟杆菌；以及药学上可接受的辅料。发明人惊喜地发现，通过对动物提供该药物组合物，能够明显抑制关节炎的发生，关节发红和肿胀明显减退，关节炎症状明显减轻，动物行动灵活，表明该药物组合物能够有效治疗或预防类风湿性关节炎或其相关疾病。In a second aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes: Bacteroides multuri; and pharmaceutically acceptable auxiliary materials. The inventors are surprised to find that by providing the pharmaceutical composition to animals, the occurrence of arthritis can be significantly inhibited, the redness and swelling of the joints can be significantly reduced, the symptoms of arthritis can be significantly reduced, and the animals can move flexibly, indicating that the pharmaceutical composition can effectively treat or Prevention of rheumatoid arthritis or related diseases.
根据本发明的实施例，所述多氏拟杆菌为Bacteroides dorei。由此，能够有效发挥治疗或预防类风湿性关节炎或其相关疾病的功效。According to an embodiment of the present invention, the Bacteroides dorei is Bacteroides dorei. Thus, the effect of treating or preventing rheumatoid arthritis or related diseases can be effectively exerted.
根据本发明的实施例，所述多氏拟杆菌为选自Bacteroides dorei DSM 17855、Bacteroides dorei JCM 13472和Bacteroides dorei BD1中的至少一种。由此，治疗或预防类风湿性关节炎或其相关疾病的效果较佳。According to an embodiment of the present invention, the Bacteroides dorei is at least one selected from Bacteroides dorei DSM 17855, Bacteroides dorei JCM 13472 and Bacteroides dorei BD1. Therefore, the effect of treating or preventing rheumatoid arthritis or related diseases is better.
根据本发明的实施例，当所述药物组合物呈固态时，所述药物组合物包含1×10-1×1020cfu/g的所述多氏拟杆菌，当所述药物组合物呈液态时，所述药物组合物包含1×10-1×1020cfu/mL的所述多氏拟杆菌。由此，抑制关节炎发生和改善关节炎症状的效果明显，如果多氏拟杆菌含量过低，治疗或预防类风湿性关节炎的效果不理想，如果多氏拟杆菌含量过高，则治疗或预防类风湿性关节炎的的效果无明显提高，造成浪费。According to an embodiment of the present invention, when the pharmaceutical composition is in a solid state, the pharmaceutical composition contains 1×10-1×1020 cfu/g of the Bacteroides multurii, and when the pharmaceutical composition is in a liquid state When, the pharmaceutical composition comprises 1×10-1×1020 cfu/mL of the Bacteroides multani. Thus, the effect of inhibiting the occurrence of arthritis and improving the symptoms of arthritis is obvious. If the content of Bacteroides dorsii is too low, the effect of treating or preventing rheumatoid arthritis is not satisfactory. The effect of preventing rheumatoid arthritis is not significantly improved, resulting in waste.
根据本发明的一个优选实施例，当所述药物组合物呈固态时，所述药物组合物包含1×104-1×1015cfu/g的所述多氏拟杆菌，当所述药物组合物呈液态时，所述药物组合物包含1×104-1×1015cfu/mL的所述多氏拟杆菌。According to a preferred embodiment of the present invention, when the pharmaceutical composition is in a solid state, the pharmaceutical composition contains 1×104 -1×1015 cfu/g of the Bacteroides multellii, when the pharmaceutical composition When the substance is in a liquid state, the pharmaceutical composition comprises 1×104 -1×1015 cfu/mL of the Bacteroides multani.
根据本发明的另一个优选实施例，当所述药物组合物呈固态时，所述药物组合物包含1×106-1×1011cfu/g，的所述多氏拟杆菌，当所述药物组合物呈液态时，所述药物组合物包含1×106-1×1011cfu/mL的所述多氏拟杆菌。According to another preferred embodiment of the present invention, when the pharmaceutical composition is in a solid state, the pharmaceutical composition contains 1×106 -1×1011 cfu/g of the Bacteroides multurii, when the When the pharmaceutical composition is in a liquid state, the pharmaceutical composition contains 1×106 -1×1011 cfu/mL of the Bacteroides multani.
根据本发明的实施例，所述药学上可接受的辅料为选自载体、赋形剂、稀释剂、润滑剂、润湿剂、乳化剂、悬浮液稳定剂、防腐剂、甜味剂以及香料中的至少一种。According to an embodiment of the present invention, the pharmaceutically acceptable adjuvant is selected from carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and spices at least one of the
根据本发明的实施例，所述药学上可接受的辅料为选自乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖、淀粉、阿拉伯胶、磷酸钙、藻酸盐、明胶、硅酸钙、细结晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁和矿物油中的至少一种。According to an embodiment of the present invention, the pharmaceutically acceptable auxiliary material is selected from lactose, glucose, sucrose, sorbitol, mannose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, fine At least one of crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.
在本发明的第三方面，本发明提供了一种药物。根据本发明的实施例，该药物包括：前面所述的药物组合物。通过对动物提供该药物，能够明显抑制关节炎的发生，关节发红和肿胀明显减退，关节炎症状明显减轻，动物行动灵活，表明该药物能够有效治疗或预防类风湿性关节炎或其相关疾病。In a third aspect of the invention, the invention provides a medicament. According to an embodiment of the present invention, the medicine includes: the aforementioned pharmaceutical composition. By providing the drug to animals, the occurrence of arthritis can be significantly inhibited, the redness and swelling of the joints can be significantly reduced, the symptoms of arthritis can be significantly reduced, and the animals can move flexibly, indicating that the drug can effectively treat or prevent rheumatoid arthritis or related diseases .
根据本发明的实施例，所述药物呈选自颗粒剂、胶囊、片剂、粉末剂、口服液、混悬液和乳剂中的至少一种制剂形式。由此，易于进行给药。According to an embodiment of the present invention, the medicine is in the form of at least one preparation selected from granules, capsules, tablets, powders, oral liquids, suspensions and emulsions. Thus, administration is facilitated.
在本发明的第四方面，本发明提供了一种食品。根据本发明的实施例，该食品包括：多氏拟杆菌；以及食品学上可接受的辅料。发明人发现，通过对动物提供该食品，能够明显抑制关节炎的发生，关节发红和肿胀明显减退，关节炎症状明显减轻，动物行动灵活，表明该食品能够有效治疗或预防类风湿性关节炎或其相关疾病。In a fourth aspect of the invention, the invention provides a food product. According to an embodiment of the present invention, the food includes: Bacteroides multuri; and food acceptable auxiliary materials. The inventor found that by providing the food to animals, the occurrence of arthritis can be significantly inhibited, the redness and swelling of the joints can be significantly reduced, the symptoms of arthritis can be significantly reduced, and the animals can move flexibly, indicating that the food can effectively treat or prevent rheumatoid arthritis or its related diseases.
需要说明的是，本发明所使用的术语“食品”的种类不受特别限制，可以为任何公知的食品，包括但不限于奶制品、饼干、糕点、饮料、保健品等。It should be noted that the type of the term "food" used in the present invention is not particularly limited, and may be any known food, including but not limited to dairy products, biscuits, cakes, beverages, health products, etc.
根据本发明的实施例，所述多氏拟杆菌为Bacteroides dorei。由此，能够有效发挥治疗或预防类风湿性关节炎或其相关疾病的功效。According to an embodiment of the present invention, the Bacteroides dorei is Bacteroides dorei. Thus, the effect of treating or preventing rheumatoid arthritis or related diseases can be effectively exerted.
根据本发明的实施例，所述多氏拟杆菌为选自Bacteroides dorei DSM 17855、Bacteroides dorei JCM 13472和Bacteroides dorei BD1中的至少一种。由此，治疗或预防类风湿性关节炎或其相关疾病的效果较佳。According to an embodiment of the present invention, the Bacteroides dorei is at least one selected from Bacteroides dorei DSM 17855, Bacteroides dorei JCM 13472 and Bacteroides dorei BD1. Therefore, the effect of treating or preventing rheumatoid arthritis or related diseases is better.
根据本发明的实施例，当所述食品呈固态时，所述食品包含1×10-1×1020cfu/g的所述多氏拟杆菌，当所述食品呈液态时，所述食品包含1×10-1×1020cfu/mL的所述多氏拟杆菌。由此，抑制关节炎发生和改善关节炎症状的效果明显，如果多氏拟杆菌含量过低，治疗或预防类风湿性关节炎的效果不理想，如果多氏拟杆菌含量过高，则治疗或预防类风湿性关节炎的的效果无明显提高，造成浪费。According to an embodiment of the present invention, when the food is in a solid state, the food contains 1×10-1×1020 cfu/g of the Bacteroides multellii, and when the food is in a liquid state, the food contains 1×10-1×1020 cfu/mL of the Bacteroides multurii. Thus, the effect of inhibiting the occurrence of arthritis and improving the symptoms of arthritis is obvious. If the content of Bacteroides dorsii is too low, the effect of treating or preventing rheumatoid arthritis is not ideal. The effect of preventing rheumatoid arthritis is not significantly improved, resulting in waste.
根据本发明的一个优选实施例，当所述食品呈固态时，所述食品包含1×104-1×1015cfu/g的所述多氏拟杆菌，当所述食品呈液态时，所述食品包含1×104-1×1015cfu/mL的所述多氏拟杆菌。According to a preferred embodiment of the present invention, when the food is in a solid state, the food contains 1×104 -1×1015 cfu/g of the Bacteroides multurii; when the food is in a liquid state, the Said food contains 1×104 -1×1015 cfu/mL of said Bacteroides multani.
根据本发明的另一个优选实施例，当所述食品呈固态时，所述食品包含1×106-1×1011cfu/g，的所述多氏拟杆菌，当所述食品呈液态时，所述食品包含1×106-1×1011cfu/mL的所述多氏拟杆菌。According to another preferred embodiment of the present invention, when the food is in a solid state, the food contains 1×106 -1×1011 cfu/g, of the Bacteroides multurii, and when the food is in a liquid state , the food contains 1×106 -1×1011 cfu/mL of the Bacteroides multani.
根据本发明的实施例，所述食品学上可接受的辅料为选自载体、赋形剂、稀释剂、润滑剂、润湿剂、乳化剂、悬浮液稳定剂、防腐剂、甜味剂以及香料中的至少一种。According to an embodiment of the present invention, the food acceptable auxiliary material is selected from carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and at least one of the spices.
根据本发明的实施例，所述食品学上可接受的辅料为选自乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖、淀粉、阿拉伯胶、磷酸钙、藻酸盐、明胶、硅酸钙、细结晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁和矿物油中的至少一种。According to an embodiment of the present invention, the food acceptable auxiliary material is selected from lactose, glucose, sucrose, sorbitol, mannose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, At least one of fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate and mineral oil.
根据本发明的实施例，所述食品呈选自固体、乳品、溶液制品、粉末制品和悬浮液制品中的至少一种制剂形式。According to an embodiment of the present invention, the food is in the form of at least one formulation selected from solids, dairy products, solution products, powder products and suspension products.
在本发明的第五方面，本发明提供了一种饲料。根据本发明的实施例，该饲料包括：多氏拟杆菌。发明人发现，通过对动物提供该饲料，能够明显抑制关节炎的发生，动物关节发红和肿胀明显减退，关节炎症状明显减轻，动物行动灵活，表明该饲料能够有效治疗或预防类风湿性关节炎或其相关疾病。In a fifth aspect of the present invention, the present invention provides a feed. According to an embodiment of the present invention, the feed includes: Bacteroides multani. The inventor found that by providing the feed to animals, the occurrence of arthritis can be significantly inhibited, the redness and swelling of the animal joints are significantly reduced, the symptoms of arthritis are significantly reduced, and the animals are flexible, indicating that the feed can effectively treat or prevent rheumatoid arthritis. inflammation or related diseases.
根据本发明的实施例，所述多氏拟杆菌为Bacteroides dorei。由此，能够有效发挥治疗或预防类风湿性关节炎或其相关疾病的功效。According to an embodiment of the present invention, the Bacteroides dorei is Bacteroides dorei. Thus, the effect of treating or preventing rheumatoid arthritis or related diseases can be effectively exerted.
根据本发明的实施例，所述多氏拟杆菌为选自Bacteroides dorei DSM 17855、Bacteroides dorei JCM 13472和Bacteroides dorei BD1中的至少一种。由此，治疗或预防类风湿性关节炎或其相关疾病的效果较佳。According to an embodiment of the present invention, the Bacteroides dorei is at least one selected from Bacteroides dorei DSM 17855, Bacteroides dorei JCM 13472 and Bacteroides dorei BD1. Therefore, the effect of treating or preventing rheumatoid arthritis or related diseases is better.
根据本发明的实施例，当所述饲料呈固态时，所述饲料包含1×10-1×1020cfu/g的所述多氏拟杆菌，当所述饲料呈液态时，所述饲料包含1×10-1×1020cfu/mL的所述多氏拟杆菌。由此，抑制关节炎发生和改善关节炎症状的效果明显，如果多氏拟杆菌含量过低，治疗或预防类风湿性关节炎的效果不理想，如果多氏拟杆菌含量过高，则治疗或预防类风湿性关节炎的的效果无明显提高，造成浪费。According to an embodiment of the present invention, when the feed is in a solid state, the feed contains 1×10-1×1020 cfu/g of the Bacteroides multellii; when the feed is in a liquid state, the feed contains 1×10-1×1020 cfu/mL of the Bacteroides multurii. Thus, the effect of inhibiting the occurrence of arthritis and improving the symptoms of arthritis is obvious. If the content of Bacteroides dorsii is too low, the effect of treating or preventing rheumatoid arthritis is not satisfactory. The effect of preventing rheumatoid arthritis is not significantly improved, resulting in waste.
根据本发明的一个优选实施例，当所述饲料呈固态时，所述饲料包含1×104-1×1015cfu/g的所述多氏拟杆菌，当所述饲料呈液态时，所述饲料包含1×104-1×1015cfu/mL的所述多氏拟杆菌。According to a preferred embodiment of the present invention, when the feed is in a solid state, the feed contains 1×104 -1×1015 cfu/g of the Bacteroides tenerii; when the feed is in a liquid state, the The feed contains 1×104 -1×1015 cfu/mL of the Bacteroides multani.
根据本发明的另一个优选实施例，当所述饲料呈固态时，所述饲料包含1×106-1×1011cfu/g，的所述多氏拟杆菌，当所述饲料呈液态时，所述饲料包含1×106-1×1011cfu/mL的所述多氏拟杆菌。According to another preferred embodiment of the present invention, when the feed is solid, the feed contains 1×106 -1×1011 cfu/g of the Bacteroides multurii, and when the feed is liquid , the feed contains 1×106 -1×1011 cfu/mL of the Bacteroides multani.
实施例1确定治疗或预防类风湿性关节炎的有益菌Example 1 Determine the beneficial bacteria for the treatment or prevention of rheumatoid arthritis
1.1样品收集和DNA提取1.1 Sample collection and DNA extraction
从北京协和医院共采集到212个志愿者的粪便、牙垢及唾液样品，样品信息见表1，其中，分为训练组和试验组。训练组包括157个志愿者，其中77个为没有治疗过的RA病例，80个为健康者(对照)。试验组中，其中34个志愿者形成17个“病例-健康”对，其中有8对有血缘关系，另外9对没有血缘关系；其他21个志愿者为DMARD(disease-modifying antirheumatic drugs)治疗RA病例。A total of 212 samples of feces, tartar and saliva were collected from Peking Union Medical College Hospital. The sample information is shown in Table 1. Among them, they were divided into training group and test group. The training group included 157 volunteers, of which 77 were untreated RA cases and 80 were healthy individuals (controls). In the test group, 34 volunteers formed 17 "case-healthy" pairs, 8 of which were related by blood, and the other 9 pairs were not related by blood; the other 21 volunteers were treated with DMARD (disease-modifying antirheumatic drugs) for RA cases.
采集粪便样品后冷冻运输并迅速转移到-80℃保存，进行DNA提取(参考Qin,J.et al.A metagenome-wide association study of gut microbiota in type 2diabetes.Nature 490,55–60(2012))。采集牙垢样品时，利用医用钳从牙齿表面刮取3μl的牙垢，转移至200μl 1x裂解缓冲液(10mM Tris,1mM EDTA，0.5％吐温20，200μg/ml蛋白酶K)中，在55℃培养2小时，然后在95℃培养10分钟，终止裂解反应，最后于-80℃保存。采集唾液样品时，取100μl样品至含100μl 2x裂解缓冲液的收集管中，同时，刮取后咽表面样品也加至该管中，后续裂解步骤与牙垢样品相同，同样于-80℃保存。牙垢样品和唾液样品经冷冻运输至深圳华大基因研究院，参照粪便样品进行DNA提取。Feces samples were collected and transported frozen and quickly transferred to -80°C for DNA extraction (refer to Qin, J. et al. A metagenome-wide association study of gut microbiota in type 2diabetes. Nature 490,55–60(2012)) . When collecting tartar samples, use medical forceps to scrape 3 μl of tartar from the tooth surface, transfer it to 200 μl 1x lysis buffer (10mM Tris, 1mM EDTA, 0.5% Tween 20, 200μg/ml proteinase K), and incubate at 55°C for 2 hours, and then incubated at 95°C for 10 minutes to terminate the lysis reaction, and finally stored at -80°C. When collecting saliva samples, take 100 μl of samples into a collection tube containing 100 μl of 2x lysis buffer. At the same time, scrape the sample of the surface of the pharynx and add it to the tube. The subsequent lysis steps are the same as that of the tartar sample, and they are also stored at -80°C. Tartar samples and saliva samples were frozen and transported to Shenzhen Huada Gene Research Institute, and DNA was extracted with reference to stool samples.
当志愿者首次入院时，按照标准程序收集表型信息。21个DMARD治疗病例的粪便样品仅包含在用来构建肠道微生物基因集的212个粪便样品之中，在本文不再进行分析。本研究得到北京协和医院和深圳华大基因研究院伦理审查委员会批准。Phenotypic information was collected following standard procedures when volunteers were first admitted. Fecal samples from 21 DMARD-treated cases were included only in the 212 stool samples used to construct the gut microbial gene set and were not analyzed in this paper. This study was approved by the ethics review committees of Peking Union Medical College Hospital and Shenzhen Huada Gene Research Institute.
表1样品信息Table 1 Sample Information
1.2宏基因组测序与组装1.2 Metagenome sequencing and assembly
利用所提取的DNA样本构建测序文库，在Illumina测序平台上进行双向(Paired-end)宏基因组测序(插入片段350bp，读长100bp)。对测序产生的数据进行过滤(quality-controlled，去除adapter污染序列、去低质量序列和去宿主基因组污染序列)，并利用SOAPdenovo软件(v2.04)进行重头组装。The extracted DNA samples were used to construct a sequencing library, and two-way (Paired-end) metagenomic sequencing (insert fragment 350bp, read length 100bp) was performed on the Illumina sequencing platform. The data generated by the sequencing was filtered (quality-controlled, removing adapter contamination sequences, removing low-quality sequences and removing host genome contamination sequences), and using SOAPdenovo software (v2.04) for de novo assembly.
粪便、牙垢和唾液样品的平均宿主基因组污染分别是0.37％、5.55％和40.85％。The average host genome contamination of feces, tartar and saliva samples was 0.37%, 5.55% and 40.85%, respectively.
1.3基因集构建1.3 Gene set construction
对于组装好的组装片段(contigs)，利用GeneMark软件(v2.7d)进行基因预测，接着利用BLAT软件进行去冗余(比对相似度(identity)在95％以上，比对的覆盖度(overlap)在90％以上，没有缺口(gaps))，对于212个粪便样品(其中包括21个DMARD治疗样品)，得到了包含3,800,011个基因的非冗余基因集；对于203个口腔样品(包括105个牙垢样品和98唾液样品)，得到了包含3,234,997个基因的非冗余基因集。利用BLAT软件将粪便样品基因集进一步补充到已公开的包含430多万个基因的肠道微生物参考基因集中(比对相似度在95％以上，比对覆盖度在90％以上，参考上述文献Qin,J.et al.2012)，最终得到了包含590多万个基因的新基因集。For the assembled assembly fragments (contigs), use GeneMark software (v2.7d) to perform gene prediction, and then use BLAT software to perform de-redundancy (the comparison similarity (identity) is above 95%, and the coverage of the comparison (overlap ) above 90%, without gaps (gaps)), for 212 stool samples (including 21 DMARD treatment samples), a non-redundant gene set containing 3,800,011 genes was obtained; for 203 oral samples (including 105 tartar samples and 98 saliva samples), a non-redundant gene set containing 3,234,997 genes was obtained. Use BLAT software to further supplement the fecal sample gene set to the published reference gene set of intestinal microorganisms containing more than 4.3 million genes (comparison similarity is above 95%, and comparison coverage is above 90%, refer to the above-mentioned literature Qin , J.et al.2012), and finally obtained a new gene set containing more than 5.9 million genes.
将高质量测序片段(reads)与肠道或口腔参考基因集进行比对，从而得到基因的相对丰度(参考上述文献Qin,J.et al.2012)。Compare the high-quality sequencing fragments (reads) with the intestinal or oral reference gene set to obtain the relative abundance of the gene (refer to the above-mentioned literature Qin, J. et al. 2012).
1.4物种分类注释与丰度计算1.4 Species classification annotation and abundance calculation
通过与IMG(v400)数据库进行比对，对预测的基因进行物种分类(参考上述文献Qin,J.et al.2012)，分别得到门水平的物种分类(比对相似度在65％以上，比对覆盖度在70％以上)，属水平的物种分类(比对相似度在85％以上)及种水平的物种分类(比对相似度在95％以上)。利用基因的相对丰度计算该物种的相对丰度(参考上述文献Qin,J.etal.2012)，并用秩和检验(Wilcoxon rank-sum test)进行统计检验(p<0.05)，确定病例与对照之间的相对丰度存在显著差异的物种。By comparing with the IMG (v400) database, the species classification of the predicted genes is carried out (refer to the above-mentioned literature Qin, J. et al. 2012), and the species classification at the phylum level is respectively obtained (the comparison similarity is above 65%, and the comparison For coverage above 70%), genus-level species classification (comparison similarity above 85%) and species-level species classification (comparison similarity above 95%). Use the relative abundance of the gene to calculate the relative abundance of the species (refer to the above-mentioned literature Qin, J.etal.2012), and use the Wilcoxon rank-sum test (Wilcoxon rank-sum test) for statistical testing (p<0.05) to determine the case and control Species with significant differences in relative abundance.
1.5宏基因组关联分析1.5 Metagenome association analysis
通过比较病例与对照的粪便微生物基因集(microbiome)，在3,110,085个基因(每个基因都至少出现在6个样品中，样品n＝157，不含表1所述21个DMARD治疗病例)中，发现83,858个基因的相对丰度具有显著差异(p<0.01,秩和检验，FDR＝0.3285)，这些基因即为标记基因。根据这些标记基因在样品中的相对丰度差异，进一步聚类，得到粪便的宏基因组连锁群(Metagenomic Linkage Group，MLG，一个MLG近似于一个物种，聚类方法参考上述文献Qin,J.et al.2012)。同样的方法，从牙垢的2,247,835个基因(每个基因都至少出现在6个样品中，样品n＝105)中，筛选出209,820个标记基因(p<0.01,秩和检验，FDR＝0.072)，聚类，得到牙垢的MLG。从唾液的2,404,726个基因(每个基因都至少出现在6个样品中，样品n＝98)中，筛选出206,399个标记基因(p<0.01,秩和检验，FDR＝0.088)，聚类，得到唾液MLG。另外，通过比较DMARD治疗前后的粪便微生物基因，从1,538,688个基因(每个基因都至少出现在6个样品中，样品n＝64)中，筛选出86158个标记基因(p<0.05,paired Wilcoxon rank-sum test,FDR＝0.912)，聚类到DMARD治疗病例的粪便MLGs中。By comparing the fecal microbial gene set (microbiome) of cases and controls, in 3,110,085 genes (each gene appears in at least 6 samples, sample n=157, excluding the 21 DMARD treatment cases described in Table 1), 83,858 genes were found to have significant differences in relative abundance (p<0.01, rank sum test, FDR=0.3285), and these genes were marker genes. According to the relative abundance difference of these marker genes in the sample, further clustering is performed to obtain the Metagenomic Linkage Group (MLG) of feces. One MLG is similar to one species. For the clustering method, refer to the above-mentioned literature Qin, J. et al .2012). In the same way, 209,820 marker genes (p<0.01, rank sum test, FDR=0.072) were screened out from 2,247,835 genes of tartar (each gene appeared in at least 6 samples, sample n=105), Clustering to get the MLG of tartar. From 2,404,726 genes in saliva (each gene appears in at least 6 samples, sample n=98), 206,399 marker genes (p<0.01, rank sum test, FDR=0.088) were screened out, and clustered to obtain Saliva-MLG. In addition, by comparing fecal microbial genes before and after DMARD treatment, 86,158 marker genes (p<0.05, paired Wilcoxon rank -sum test, FDR=0.912), clustered into fecal MLGs of DMARD-treated cases.
根据MLGs组成基因的分类及相对丰度，进行MLGs物种分类和构建MLGs相对丰度谱(参考上述文献Qin,J.et al.2012)。若要分类到种的水平，需要满足MLGs中90％的基因可以比对到该物种的基因组上(比对相似度在95％以上，比对覆盖度在70％以上)。若要分类到属水平，则需要MLGs中80％的基因，无论是在DNA序列还是蛋白质序列，与该属的相似度都要高于80％。根据所有样品丰度之间的Kendall’s相关性(regardless ofcase-control status)，可以将MLGs进行进一步地聚类。肠道MLG、唾液MLG、牙垢MLG和DMARD治疗相关MLG的物种分类结果分别见表2、表3、表4、表5。According to the classification and relative abundance of the constituent genes of MLGs, the species classification of MLGs and the relative abundance spectrum of MLGs were constructed (refer to the above literature Qin, J. et al. 2012). To classify to the species level, it is necessary to satisfy that 90% of the genes in MLGs can be compared to the genome of the species (the alignment similarity is above 95%, and the alignment coverage is above 70%). To classify to the genus level, 80% of the genes in MLGs need to have more than 80% similarity with the genus, whether in DNA sequence or protein sequence. MLGs can be further clustered according to the Kendall's correlation (regardless of case-control status) between the abundances of all samples. The species classification results of intestinal MLG, salivary MLG, tartar MLG, and DMARD treatment-related MLG are shown in Table 2, Table 3, Table 4, and Table 5, respectively.
表2肠道MLG的物种分类信息Table 2 Species classification information of intestinal MLG
表3唾液MLG的物种分类信息Table 3 Taxonomic information of salivary MLG species
表4牙垢MLG的物种分类信息Table 4 Species classification information of tartar MLG
表5DMARD治疗相关MLG的物种分类信息Table 5 Species classification information of DMARD treatment-related MLG
实施例2动物实验验证Embodiment 2 animal experiment verification
为了验证实施例1在表2、表3、表4、表5确定治疗或预防类风湿性关节炎的有益菌，发明人对每个菌种进行动物实验，利用该菌种可获得的菌株验证其功效。In order to verify that Example 1 determines the beneficial bacteria for the treatment or prevention of rheumatoid arthritis in Table 2, Table 3, Table 4, and Table 5, the inventor conducts animal experiments for each bacterial species, and uses the available bacterial strain verification of the bacterial species its efficacy.
实验方法experimental method
1.造模及给药1. Modeling and administration
6～8周龄SPF(Specific pathogen free)级雄性DBA/1J小鼠，购于中国科学院上海实验动物中心，饲养于IVC(individual ventilated cage)鼠笼系统(购自苏州冯氏实验动物设备有限公司)，室内保持温度22～24℃，湿度40％～60％，每日光照12h，自由进食及饮水。适应性饲养2周后随机分为5组(模型对照组、菌剂1组、菌剂2组、菌剂3组、正常对照组)，每组12只。Bacteroides dorei DSM 17855(菌剂1)购自DSMZ(Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH)；Bacteroides dorei JCM 13472(菌剂2)购自JCM(Japan Collection ofMicroorganisms)；Bacteroides dorei BD1(菌剂3)来源于丹麦欧登塞大学医院临床微生物系(Department of Clinical Microbiology,Odense University Hospital,Odense,Denmark)。于改良PYG培养液(DSMZ Medium104)中，37℃厌氧培养1-2天，经16SrDNA测序鉴定无误后开始实验。菌株信息见表6。6-8 weeks old SPF (Specific pathogen free) grade male DBA/1J mice were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences and raised in the IVC (individual ventilated cage) mouse cage system (purchased from Suzhou Fengshi Experimental Animal Equipment Co., Ltd. ), the indoor temperature was kept at 22-24°C, the humidity was 40%-60%, the light was 12 hours a day, and food and water were free to eat. After 2 weeks of adaptive feeding, they were randomly divided into 5 groups (model control group, bacterial agent 1 group, bacterial agent 2 group, bacterial agent 3 group, and normal control group), with 12 rats in each group. Bacteroides dorei DSM 17855 (bacteria agent 1) was purchased from DSMZ (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH); Bacteroides dorei JCM 13472 (bacteria agent 2) was purchased from JCM (Japan Collection of Microorganisms); 3) Sourced from Department of Clinical Microbiology, Odense University Hospital, Odense, Denmark. In the improved PYG medium (DSMZ Medium104), cultured anaerobically at 37°C for 1-2 days, and the experiment was started after the 16S rDNA sequencing confirmed that it was correct. See Table 6 for strain information.
表6菌株信息Table 6 strain information
参考S.Yoshino的方法(S.Yoshino,E.Sasatomi,M.Ohsawa,Bacterial lipopolysaccharideacts as an adjuvant to induce autoimmune arthritis in mice,2000)的造模方法，简言之：Refer to the modeling method of S.Yoshino's method (S.Yoshino, E.Sasatomi, M.Ohsawa, Bacterial lipopolysaccharideacts as an adjuvant to induce autoimmune arthritis in mice, 2000), in short:
自实验第1天起，菌剂1组(菌剂1+CIA/LPS)、菌剂2组(菌剂2+CIA/LPS)、菌剂3组(菌剂3+CIA/LPS)分别以500μl/50g.BW剂量，灌胃菌1、菌2、菌3新鲜培养液(109-1010cfu/ml)，模型对照组(M+CIA/LPS)和正常对照组(M+AA+PBS)分别灌胃等体积无菌培养液，隔天给药28天。在第1天、第21天，利用II型胶原(CIA)结合脂多糖(LPS)诱导3个模型组(菌剂1组、菌剂2组及菌剂3组)及模型对照组小鼠关节炎的发生。具体操作为：将100mg II型胶原蛋白(取自小牛关节软骨,Funakoshi Co)溶于100ml 5mM乙酸(AA)，4℃搅拌过夜，腹腔注射小鼠。随后取E.coli 011:B4的脂多糖(上海生物制品研究所)1μg，溶于0.1ml磷酸缓冲液(PBS)，同样经腹腔注射给药。正常对照组在相同时间点经同体积的5mM乙酸及PBS缓冲液腹腔注射。每天观察小鼠足爪状态，每周对关节炎指数进行评分。于实验开始后35天，每组随机选取6只处死，取脾脏细胞进行后续实验。剩余小鼠继续观察关节炎病程进展情况并在实验开始后50天颈椎脱臼处死。From the first day of the experiment, bacterial agent 1 group (bacterial agent 1+CIA/LPS), bacterial agent 2 group (bacterial agent 2+CIA/LPS), bacterial agent 3 group (bacterial agent 3+CIA/LPS) respectively 500μl/50g.BW dose, intragastric administration of bacteria 1, bacteria 2, bacteria 3 fresh culture solution (109-1010cfu/ml), model control group (M+CIA/LPS) and normal control group (M+AA+PBS) respectively Gastrointestinal administration of equal volume of sterile culture solution for 28 days every other day. On the 1st day and the 21st day, use type II collagen (CIA) combined with lipopolysaccharide (LPS) to induce three model groups (microbial agent 1 group, bacterial agent 2 group and bacterial agent 3 group) and the model control group mice joints The occurrence of inflammation. The specific operation was as follows: 100mg type II collagen (taken from calf articular cartilage, Funakoshi Co) was dissolved in 100ml 5mM acetic acid (AA), stirred overnight at 4°C, and intraperitoneally injected into mice. Then take 1 μg of E.coli 011:B4 lipopolysaccharide (Shanghai Institute of Biological Products), dissolve it in 0.1ml phosphate buffered solution (PBS), and administer it by intraperitoneal injection as well. The normal control group was intraperitoneally injected with the same volume of 5mM acetic acid and PBS buffer at the same time point. The state of the paws of the mice was observed every day, and the arthritis index was scored every week. 35 days after the start of the experiment, 6 animals in each group were randomly selected to be sacrificed, and spleen cells were collected for subsequent experiments. The remaining mice continued to observe the progression of arthritis and were killed by cervical dislocation 50 days after the start of the experiment.
2.关节炎评价2. Arthritis evaluation
利用肉眼根据红斑的增加以及关节周围组织水肿情况对关节炎的严重程度进行宏观评分得到关节炎指数，具体评分标准描述如下：0：没有关节炎的迹象；1：1个关节肿胀和/或发红；2：2个关节肿胀和/或发红；3：大于2个关节肿胀和/或发红；4：整个足爪发生严重关节炎症。每只小鼠关节炎指数为每只动物4爪得分之和。Macroscopically score the severity of arthritis according to the increase of erythema and tissue edema around the joint to obtain the arthritis index. The specific scoring criteria are described as follows: 0: no signs of arthritis; 1: 1 joint swelling and/or swelling Red; 2: swelling and/or redness of 2 joints; 3: swelling and/or redness of more than 2 joints; 4: severe joint inflammation of the entire paw. The arthritis index of each mouse is the sum of the scores of the 4 paws of each animal.
3.菌剂1、菌剂2和菌剂3对胶原诱导的关节炎小鼠脾细胞体外增殖的影响3. Effects of bacterial agent 1, bacterial agent 2 and bacterial agent 3 on the proliferation of spleen cells in mice with collagen-induced arthritis in vitro
随机选取模型组(菌剂1组、菌剂2组及菌剂3组)、模型对照组和正常对照组每组各6只小鼠，常规分离脾细胞。后续操作参考赵晓辉等的方法(塞隆骨提取物对牛II型胶原诱导的小鼠关节炎的治疗作用及机制研究，赵晓辉，岳会兰，梅丽娟，邵赞，陶燕铎，2008年5月)：用含10％小牛血清的RPMI 1640培养液将细胞浓度调成4×106cell/ml。于96孔板中每孔加入100μl细胞悬液，100μl胶原蛋白抗原。细胞于含5％CO2的37℃培养箱中培养，培养结束前8h每孔加入25μl 3H-胸腺嘧啶核苷酸。继续培养至实验结束。用细胞收集仪将细胞收集至玻璃纤维膜上，加入闪烁液后于β计数仪上读取细胞DNA中3H-胸腺嘧啶核苷酸量，以每分钟脉冲数(counts per minute，cpm)表示细胞增殖情况。Randomly select model group (microbial agent 1 group, bacterial agent 2 group and bacterial agent 3 group), model control group and normal control group with 6 mice in each group, and routinely isolate splenocytes. Follow-up operations refer to the method of Zhao Xiaohui et al. (Study on Therapeutic Effect and Mechanism of Cylon Bone Extract on Bovine Type II Collagen-Induced Arthritis in Mice, Zhao Xiaohui, Yue Huilan, Mei Lijuan, Shao Zan, Tao Yanduo, May 2008 ): adjust the cell concentration to 4×106 cell/ml with RPMI 1640 culture medium containing 10% calf serum. Add 100 μl of cell suspension and 100 μl of collagen antigen to each well of a 96-well plate. The cells were cultured in a 37°C incubator containing 5% CO2 , and 25 μl3 H-thymidine nucleotide was added to each well 8 hours before the end of the culture. Continue to cultivate until the end of the experiment. Collect the cells on the glass fiber membrane with a cell harvester, add scintillation fluid, and read the amount of3 H-thymidine nucleotides in the cell DNA on a β counter, expressed in counts per minute (cpm) cell proliferation.
4.菌剂1、菌剂2和菌剂3对关节炎小鼠炎性细胞因子的影响4. Effects of bacterial agent 1, bacterial agent 2 and bacterial agent 3 on inflammatory cytokines in arthritis mice
常规方法分离脾细胞，用含10％小牛血清的RPMI 1640培养液将细胞浓度调成4×106cell/ml，以脾细胞悬液1ml/孔接种于24孔组织培养板中，其中含1mM谷氨酰胺，100U/ml青霉素，100mg/ml链霉素，5×10-5M 2-巯基乙醇及1％热灭活自体血清。48h后，收集上清液于-70℃保存，利用ELISA试剂盒(购于上海酶联生物科技有限公司)检测其中细胞因子IL-12，IFN-γ，TNF-α和IL-10水平。Spleen cells were isolated by conventional methods, and the cell concentration was adjusted to 4×106 cell/ml with RPMI 1640 culture medium containing 10% calf serum, and 1 ml/well of spleen cell suspension was inoculated in a 24-well tissue culture plate, which contained 1mM glutamine, 100U/ml penicillin, 100mg/ml streptomycin, 5×10-5 M 2-mercaptoethanol and 1% heat-inactivated autologous serum. After 48 hours, the supernatant was collected and stored at -70°C, and the levels of cytokines IL-12, IFN-γ, TNF-α and IL-10 were detected using ELISA kits (purchased from Shanghai Enzyme Biotechnology Co., Ltd.).
实验结果Experimental results
1.菌剂1、菌剂2和菌剂3对小鼠关节炎发病的影响。1. Effects of bacterial agent 1, bacterial agent 2 and bacterial agent 3 on the pathogenesis of arthritis in mice.
菌剂1、菌剂2和菌剂3对小鼠关节炎发病的影响的宏观评分结果见表7和图1。由实验结果可知，菌剂1、菌剂2和菌剂3给药4周后，相比模型对照组，能够明显抑制CIA小鼠关节炎的发生，小鼠足肿胀明显减退，关节炎症状明显减轻，小鼠行动较灵活。See Table 7 and Figure 1 for the macroscopic scoring results of the effects of bacterial agent 1, bacterial agent 2 and bacterial agent 3 on the onset of arthritis in mice. It can be seen from the experimental results that after 4 weeks of administration of bacterial agent 1, bacterial agent 2 and bacterial agent 3, compared with the model control group, the occurrence of arthritis in CIA mice can be significantly inhibited, the swelling of the mice's feet is significantly reduced, and the symptoms of arthritis are obvious. Lighten, the mice move more flexibly.
表7菌剂1、菌剂2和菌剂3对小鼠关节炎发病的影响结果(每组6只小鼠)Table 7 bacterial agent 1, bacterial agent 2 and microbial agent 3 are to the impact result of mouse arthritis pathogenesis (every group of 6 mice)
2.菌剂1、菌剂2和菌剂3对关节炎小鼠脾细胞体外增殖的影响2. Effects of bacterial agent 1, bacterial agent 2 and bacterial agent 3 on the proliferation of splenocytes in arthritis mice in vitro
菌剂1、菌剂2和菌剂3对关节炎小鼠脾细胞体外增殖的影响结果见表8和图2，其中，用相同字母标注表示两组间数值经Tukey’s Test检测差异不显著(p>0.05)。由试验结果可知，通过灌服菌剂1、菌剂2和菌剂3，在加抗原的情况下，关节炎小鼠脾细胞体外经抗原刺激后的增殖反应与模型对照组相比明显降低，在不加抗原的条件下，各模型组也显示了不同程度的脾细胞增殖抑制作用。See Table 8 and Figure 2 for the effects of bacterial agent 1, bacterial agent 2 and bacterial agent 3 on the in vitro proliferation of arthritic mice splenocytes, where the same letters indicate that the values between the two groups are not significantly different by Tukey's Test (p >0.05). From the test results, it can be seen that by gavage with bacterial agent 1, bacterial agent 2 and bacterial agent 3, in the case of adding antigen, the proliferative response of splenocytes in arthritis mice after antigen stimulation in vitro was significantly lower than that of the model control group, Under the condition of not adding antigen, each model group also showed different degrees of splenocyte proliferation inhibition.
表8菌剂1、菌剂2和菌剂3对关节炎小鼠脾细胞体外增殖的影响(每组6只小鼠)Table 8 Effects of bacterium agent 1, bacterium agent 2 and bacterium agent 3 on spleen cell proliferation in vitro of arthritis mice (6 mice in each group)
3.菌剂1、菌剂2和菌剂3对关节炎小鼠细胞因子的影响3. Effects of bacterial agent 1, bacterial agent 2 and bacterial agent 3 on cytokines in arthritis mice
菌剂1、菌剂2和菌剂3对关节炎小鼠细胞因子的影响结果见表9和图3，其中，相同字母标注表示两组间数值经Tukey’s Test检测差异不显著(p>0.05)。由实验结果可知，通过ELISA检测可以发现，与正常对照组比较，3个模型组小鼠细胞抑炎因子IL-10分泌显著减少(p<0.05)，炎症因子IL-12和TNF-α分泌显著增多(p<0.05)，而炎症因子IFN-γ分泌没有显著变化(p>0.05)；与模型对照组比较，模型组小鼠细胞IL-10分泌显著增多(p<0.05)，IL-12、IFN-γ、TNF-α分泌显著减少(p<0.05)，且3个模型组之间无显著差异(p>0.05)。说明菌剂1、菌剂2和菌剂3灌胃能够抑制脾细胞培养上清中炎症因子IL-12、IFN-γ、TNF-α的分泌，并提高抑炎因子IL-10的分泌，显示菌剂1、菌剂2和菌剂3对关节炎小鼠脾细胞免疫功能亢进有明显抑制作用。See Table 9 and Figure 3 for the effects of Bacteria 1, Bacteria 2 and Bacteria 3 on cytokines in mice with arthritis, where the same letters indicate that the values between the two groups are not significantly different by Tukey's Test (p>0.05) . From the experimental results, it can be seen by ELISA that, compared with the normal control group, the secretion of the anti-inflammatory factor IL-10 in the mice in the three model groups was significantly reduced (p<0.05), and the secretion of the inflammatory factors IL-12 and TNF-α was significantly reduced. increased (p<0.05), but there was no significant change in the secretion of inflammatory factor IFN-γ (p>0.05); compared with the model control group, the secretion of IL-10 in the model group was significantly increased (p<0.05), IL-12, The secretion of IFN-γ and TNF-α decreased significantly (p<0.05), and there was no significant difference among the three model groups (p>0.05). It shows that intragastric administration of bacterial agent 1, bacterial agent 2 and bacterial agent 3 can inhibit the secretion of inflammatory factors IL-12, IFN-γ, and TNF-α in the culture supernatant of splenocytes, and increase the secretion of anti-inflammatory factor IL-10, showing Bacterial agent 1, bacterium agent 2 and bacterium agent 3 had obvious inhibitory effect on splenocyte immune hyperfunction in arthritis mice.
表9菌剂1、菌剂2和菌剂3对关节炎小鼠细胞因子的影响(每组6只小鼠)The influence of table 9 bacterial agent 1, bacterial agent 2 and bacterial agent 3 on cytokines in arthritis mice (6 mice in every group)
结论in conclusion
上述结果表明，在CIA小鼠类风湿性关节炎模型上，菌剂1、菌剂2和菌剂3能够明显抑制小鼠关节炎的发病，减轻CIA小鼠的相关症状，显示出较好的抗炎作用。The above results show that on the CIA mouse rheumatoid arthritis model, bacterial agent 1, bacterial agent 2 and bacterial agent 3 can significantly inhibit the onset of arthritis in mice, alleviate the relevant symptoms of CIA mice, and show a better effect. Anti-inflammatory effect.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the described specific features, structures, materials or characteristics may be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples and features of different embodiments or examples described in this specification without conflicting with each other.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, those skilled in the art can make the above-mentioned The embodiments are subject to changes, modifications, substitutions and variations.
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| CN201410522410.2ACN104546942A (en) | 2014-09-30 | 2014-09-30 | Application of bacteroides dorei in treating or preventing rheumatoid arthritis or related diseases thereof |
| HK15110138.5AHK1209347A1 (en) | 2014-09-30 | 2015-10-15 | Use of bacteroides dorei in the treatment or prevention of rheumatoid arthritis or related diseases |
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| CN201410522410.2APendingCN104546942A (en) | 2014-09-30 | 2014-09-30 | Application of bacteroides dorei in treating or preventing rheumatoid arthritis or related diseases thereof |
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| CN104546942A (en) | Application of bacteroides dorei in treating or preventing rheumatoid arthritis or related diseases thereof | |
| CN104546940A (en) | Application of common bacteroides in treatment or prevention of rheumatoid arthritis or related diseases thereof | |
| CN104546932A (en) | Application of bacteroides ovatus in treating or preventing rheumatoid arthritis or related diseases thereof | |
| CN104546933A (en) | Application of bacteroides caccae in treatment or prevention of rheumatoid arthritis or related diseases thereof | |
| CN104546935A (en) | Application of bacteroides thetaiotaomicron in treating or preventing rheumatoid arthritis or related diseases thereof | |
| CN104546934A (en) | Application of parabacteroides merdae in treating or preventing rheumatoid arthritis or related diseases | |
| CN104546945A (en) | Application of bifidobacterium bifidum in treating or preventing rheumatoid arthritis or related diseases thereof | |
| CN104546930A (en) | Application of haemophilus parainfluenzae in treating or preventing rheumatoid arthritis or related diseases | |
| CN104546938A (en) | Application of extremely giant megamonas in treatment or prevention of rheumatoid arthritis or related diseases thereof | |
| CN104546944A (en) | Application of lactococcus lactis in treating or preventing rheumatoid arthritis or related diseases thereof | |
| CN104546947A (en) | Application of lactobacillus crispatus in treating or preventing rheumatoid arthritis or related diseases | |
| CN104546939A (en) | Application of dialister invisus in treating or preventing rheumatoid arthritis or related diseases | |
| CN104546943A (en) | Application of alistipes shahii in treating or preventing rheumatoid arthritis or related diseases | |
| CN104546936A (en) | Application of speckled prevotella in treatment or prevention of rheumatoid arthritis or related diseases thereof | |
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| CN104546931A (en) | Application of sutterella wadsworthensis in treating or preventing rheumatoid arthritis or related diseases thereof | |
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