基于肠道微生态及黏膜免疫解析双歧杆菌黏附性差异及其对便秘的影响

Constipation, which seriously affects living quality of the people, is a common gastrointestinal disease. Studies have shown that changes in the intestinal flora in the constipation group mainly involve a decrease in Bifidobacteria and an increase in pathogenic bacteria. Therefore, supplementation with probiotics has become a new method to treat constipation. Previous studies have demonstrated that the relief of Bifidobacteria for constipation may be related to adhesion properties of specialized strains, however, the underlying mechanisms is still unknown. Based on this, whole genome sequencing and comparative study will be conducted to investigate Bifidobacteria with different adhesion abilities to explore adhering-associated genes. And then mutant strain will be constructed and combined with animal models to investigate the change of gut micro-ecology and mucosal immunity. Meta-genome sequencing, GC-MS and flow cytometry will be adopted to detect the influence of wide type and mutant strains on gut microbiota, SCFAs, the numbers of CD4+CD25+ Foxp3+ Treg in lamina propria as well as immunity associated cytokines (IL-1β，IL-6，IL-10，IL-17A，TNF-α，TGF-β1，IFN-α). Finally, pseudo germ-free mice treated with wild type strains or mutant strains will be used to verify the change of functional microbiota and SCFAs. The results will illustrate the association between the adhesion properties and constipation relief of Bifidobacteria, and finally provide theoretical basis and guidance for the investigation and application of Bifidobacteria probiotics.

便秘是一种常见的胃肠道疾病，严重影响人们的生活质量。研究表明，便秘患者肠道菌群严重失调，双歧杆菌等益生菌减少，致病菌增多。体外补充益生菌可改善菌群失调，缓解便秘。前期研究表明双歧杆菌缓解便秘可能与菌株黏附性有关，但机制尚不明确。本项目针对已有的具有不同黏附性的双歧杆菌，对其进行全基因测序并比对，寻找黏附相关基因，构建突变株；应用动物模型，通过宏基因组测序，GC-MS及流式细胞术等，检测野生株和突变株对肠道菌群，SCFAs，结肠固有层Treg细胞数目以及相关免疫因子（IL-1β，IL-6，IL-10，IL-17A，TNF-α，TGF-β1，IFN-α）的影响，应用“伪无菌”鼠对野生株和突变株处理后的功能菌群及SCFAs进行验证。从肠道微生态及黏膜免疫方面深入解析双歧杆菌缓解便秘的机制。研究结果揭示了双歧杆菌黏附性与便秘缓解之间的相关性，为双歧杆菌微生态制剂的研究与应用提供理论依据和指导。

便秘是一种常见的，高发病率的，病因复杂的胃肠道疾病。目前用于治疗便秘的药物或多或少都存在口感差、作用弱、毒副作用大、患者适应性差、易产生药物依赖等问题。因此寻求一种安全、有效的药物替代品迫在眉睫。本项目通过全基因比对及基因敲除手段分析了双歧杆菌参与肠上皮黏附的功能基因。针对功能基因保守序列设计特异性扩增引物，获得的功能基因的片段克隆至pGhost 载体，通过转化、筛选、鉴定得到 黏附功能基因定向敲除的菌株。并通过细胞黏附模型及便秘小鼠验证该基因在双歧杆菌肠道定植以及便秘缓解中的作用。最终找出决定双歧杆菌缓解便秘的关键功能基因。通过宏基因组测序技术测定小鼠粪便菌群的组成。应用本课题组独有的双歧杆菌鉴定到种的引物，从种水平上探索黏附差异性双歧杆菌对便秘小鼠粪便菌群结构的影响。通过GC-MS技术测定小鼠粪便内SCFAs的含量，探索具有黏附差异的双歧杆菌对小鼠粪便SCFAs的影响。通过流式细胞术测定小鼠肠道内直接接触菌的固有层淋巴细胞中CD4+CD25+ Foxp3+ Treg细胞数目的变化情况，以及用ELISA试剂盒测定相关免疫因子（IL-1β，IL-6，IL-10，IL-17A，TNF-α，TGF-β1，IFN-α）和卵清蛋白（OVA）的变化情况，从黏膜免疫方面探索黏附差异性双歧杆菌缓解便秘的机制。应用“伪无菌”鼠验证便秘相关功能菌群及SCFAs。通过将便秘恢复组和便秘组小鼠肠道菌群及不同SCFAs（乙酸，丙酸，丁酸和这三者的混酸）移植到“伪无菌”鼠（使用广谱抗生素降低BALB/c小鼠体内肠道微生物负荷，建立“伪无菌”鼠模型）肠道中，以粪便含水量、小肠推进率以及首粒排黑便时间作为评价便秘的指标，验证所得到的功能菌群及SCFAs是否是引起便秘的原因。同时测定直接与菌接触的结肠固有层中CD4+CD25+ Foxp3+ Treg细胞数目，建立功能菌群-肠运动-免疫之间以及SCFAs-肠运动-免疫的相关性，从肠道菌群及SCFAs方面解析双歧杆菌对便秘的影响。