骨桥蛋白在肠道微生态调节肝损伤中的作用及机制研究

Recently, a close relationship between intestinal microflora and the development of liver diseases has been reported. For optimizing the treatment of liver diseases, it is an urgent need to illustrate the immune mechanism of liver disease influenced by intestinal microflora. Osteopontin (OPN), a cytokine, plays an important role in many infectious diseases. Our previous study found that administration of intestinal pathogenic bacteria would aggravate liver injury and increase endotoxin levels as well as enhance the level of OPN in liver in mice. Thus, we speculated that OPN may be the key of intestinal microflora influencing liver injury. In this study, antibiotics and intestinal pathogens are administrated to induce the imbalance of intestinal microflora in ConA-induced hepatitis animal models. Firstly, we observe the influences of different gut microflora imbalance on liver injury. Furthermore, a series of in vivo and in vitro experimental techniques (including 16SrRNA gene sequencing, DGGE fingerprint, quantitative PCR, flow cytometry, cell purification and cultivation) are used to investigate the distribution and activation of immune cells during the development of hepatitis after intestinal microflora imbalance, and illustrate the relationship and mechanism between intestinal microflora changes and OPN production in liver. Finally, feces and blood samples from different stages hepatitis patients were collected to verify the relationships between intestinal microflora and liver injury from animal experiments. In short, this study may provide a novel theory in the immune mechanism of intestinal microflora influencing the development of liver diseases, and a creative idea on clinical treatment of liver diseases.

肠道微生态与肝病发生发展存在密切关系。阐明肠道微生态调节肝病的免疫机制是优化肝病治疗的迫切需要。骨桥蛋白（OPN）作为一种细胞因子, 在肝病发展过程中发挥重要作用。我们前期实验发现肠道致病菌干预造成小鼠肝损伤加重，肝脏及肠道中OPN水平明显增高，因此我们推测OPN可能是肠道微生态调节肝损伤的关键。本课题首先利用肠道致病菌、抗生素、益生菌干预的肝炎动物模型，检测各种肠道微生态失衡对肝损伤的影响。其次，通过流式细胞术、16SrRNA基因测序、DGGE指纹谱、定量PCR、细胞纯化、培养等技术进行体内、体外实验，研究不同肠道微生态失衡对肝炎发病过程中OPN表达的影响，了解肠道菌群通过OPN影响肠道、肝脏免疫细胞活化的作用机制。最后，利用肝炎患者样本验证动物实验得出的肠道菌群失衡引起肝损伤与OPN的关系。本课题的实施，将为阐明肠道微生态影响肝病发展的免疫学机制，寻找有效的分子治疗靶点提供理论基础。

肠道微生态与多种肝病的发生发展密切相关，但是肠道微生态与肝病的因果关系及相关机制仍未完全阐明。骨桥蛋白作为一种细胞因子,在多种肝病的发生发展中发挥关键作用。然而，骨桥蛋白是否与肠道微生态相互作用共同加重肝病的发展，在本课题实施前未见报道。阐明肠道微生态在肝病中调节机制是优化肝病治疗的迫切需要。本课题已经按照研究计划进行了肠道微生态失衡在肝损伤过程中骨桥蛋白作用的研究，体内及细胞层面揭示了肝损伤时骨桥蛋白对肠道微生态的影响，以及肠道微生态失衡通过活化肠道及肝脏巨噬细胞加重肝损伤的机制，并用多种肝损伤模型研究骨桥蛋白在肠道微生态调控肝损伤中的作用。我们发现无论在肝炎、肝纤维化加重过程中以及高脂饮食引起的脂肪肝等肝损伤过程中骨桥蛋白都发挥了重要作用，骨桥蛋白可以通过影响肠黏膜产生粘附因子的数量而影响菌群的定植，并且骨桥蛋白也可以通过影响特定细菌的生长而改变肠道菌群的结构，引起细菌移位，活化肝脏巨噬细胞进一步加重肝损伤；在肝损伤的过程中，由于肝肠循环的存在，引起肠道菌群的失调，肠道菌群失调可以导致骨桥蛋白水平的升高，大量的骨桥蛋白到达肝脏，进而诱发免疫细胞杀伤肝细胞作用的增强，引起肝损伤的加重。慢乙肝患者进展到肝纤维化的过程中骨桥蛋白被凝血酶切割，暴露出新的整合素结合位点，与整合素α4和α9结合诱导星形细胞通过MAPK激酶/NF-κb信号通路促进胶原蛋白的产生加重肝纤维化，另外肝纤维化加重过程中，肠道微生态失衡表现为粪球菌增多并移位到肝脏，促进肝脏、肠道的巨噬细胞活化，进一步引起星状细胞活化加重肝损伤。在肠道菌群紊乱和肝损伤加重过程中骨桥蛋白发挥了重要作用，阻断骨桥蛋白的功能可作为预防和治疗肝损伤的新靶点。在本课题实施过程中发表论文21篇，授权发明专利5项，作为主要成员获得国家科技进步特等奖1项。多名研究生参与课题的实施，其中博士研究生3名和硕士研究生1名获得国家奖学金。