减毒肺炎链球菌呼吸道定植诱导的重度慢阻肺动物模型建立及其机制研究

Chronic obstructive pulmonary disease (COPD) is characterized by an enhanced chronic inflammatory response in the airways, and so far is short of effective treatment ways. Improved the diseases knowledge is disconnected with old animal models, which is lead to the biggest handicaps on studies of drug development and disease mechanism. Lacking the sustainable airway inflammation is the key problem to build the severe COPD models. Our previous research has shown that airway inflammation was kept going on by streptococcus pneumoniae in cigarette smoking-exposure mild COPD models. So we imagine that the severe COLD animal models will has the potential to build based on the "vicious-circle theory" between bacterial airway colonization and COPD disease progression, which is consistent with the COPD disease clinical course. Based on the previous research, in this project, we plan build the severe COLD animal models by combining cigarette smoking-exposure and knockout streptococcus pneumoniae (ΔD39)colonization in lower airway, besides, the mechanism will be explained by analyzingΔD39 strain colonization susceptibility after stopping smoking and the sustainability airway innate immune response and Th17/Treg immune imbalance. These work are all in order to build good severe COLD animal models, that are agreed with COPD disease clinical progression and are suitable for the studies of drug development and disease mechanism.

慢性阻塞性肺疾病（慢阻肺）是一种迄今缺乏有效治疗药物的慢性气道炎症性疾病。陈旧动物模型与临床慢阻肺认识更新之间的严重脱节成为药物研发和疾病机制深入研究的阻碍。“缺乏与临床相符的可持续进展的气道炎症”是目前难于构建重度、极重度慢阻肺模型的关键难题。申请人前期研究发现肺炎链球菌的呼吸道定植可以维持香烟导致的慢阻肺气道炎症的可持续进展。我们设想：利用自然病程中细菌感染/定植与慢阻肺进展间的“恶性循环假说”，有望建立与临床进程相一致的重度、极重度慢阻肺模型。本项目拟在前期研究基础上，创新性联合细菌与吸烟两大因素，构建减毒肺炎链球菌ΔD39(ply基因敲除)下呼吸道定植联合香烟暴露诱导重度、极重度慢阻肺模型，并检测分析戒烟后ΔD39定植易感性、气道固有免疫反应可持续性和Th17/Treg的免疫失衡，阐释建模机制。为慢阻肺药物的研发和疾病机制深入研究提供与临床发病机制一致的重度、极重度慢阻肺动物模型。

慢性阻塞性肺疾病（慢阻肺）是一种迄今缺乏有效逆转治疗药物的慢性气道炎症性疾病。陈旧动物模型与临床慢阻肺认识更新之间的严重脱节成为药物研发和疾病机制深入研究的阻碍。“缺乏与临床相符的可持续进展的气道炎症”是难以构建重度慢阻肺模型的关键难题。本项目首次在慢阻肺疾病基础研究中引入与真实临床相符的潜在致病菌下呼吸道定植联合香烟暴露两种因素，于24周通过检测小鼠肺功能、肺组织病理、肺泡灌洗液细胞分类计数检测、肺泡灌洗液相关细胞因子检测等一系列病理生理指标，证实成功构建了与真实临床高度一致的减毒肺炎链球菌ΔD39(ply基因敲除)下呼吸道定植联合香烟暴露诱导的稳定的重度慢阻肺模型，具备（1）肺泡灌洗液中检出持续存在的气道慢性炎症；（2）肺功能提示FEV0.1%下降到50%以下，FEV0.1/FVC 下降到70%以下，FRC 和Cst 显著升高；（3）肺组织病理呈现小气道及肺血管重塑纤维化改变和全小叶型的肺泡病理改变。分析重度慢阻肺小鼠模型下呼吸道肺泡灌洗液炎症因子、细胞因子变化情况，证实了烟雾暴露启动气道的固有免疫反应，而以肺炎链球菌为代表的潜在致病菌进一步诱发维持了Th17/Treg的免疫失衡，为重度慢阻肺慢性气道炎症的启动和维持放大提供了免疫失衡学说依据。进一步重度慢阻肺小鼠下呼吸道微生态分析及下呼吸道机会致病菌易感性研究发现：重度慢阻肺模型中下呼吸道细菌多样性及丰富度明显降低，优势菌属发现改变，对潜在致病菌的易感性增加，揭示了中重度慢阻肺微生态改变及细菌易导致AECOPD的可能机制。综上，项目的顺利完成为慢阻肺药物的研发和疾病机制深入研究提供与临床发病机制一致的重度慢阻肺动物模型实验平台，阐释了部分慢阻肺疾病进展的可能机制。