microRNA在豚鼠肺泡II型细胞抗结核免疫反应中的作用机制

Tuberculosis（TB） is a chronic, progressive infectious pulmonary disease that threats to the health in human and animals, which is caused by the infection of Mycobacterium tuberculosis (Mtb). Though both pulmonary macrophages and alveolar Type II cells (AEC II) are the first cell types encounter the pathogen, the functions of AEC II in immune responses against Mtb infection remain unclear. microRNAs have been evidenced their capacity in regulating crucial immune signals via various mechanisms, together with the facts of that the guinea pig is sensitive to Mycobacteria and a reliable animal model for understanding the immune mechanism against mycobacteria infection, as well as our primary findings in pulmonary immune signals analysis and AEC II cells isolation and culture from guinea pig, here we propose to identify and compare the microRNA expression profiles of guinea pig AEC II in response to Mycobacteria (BCG) infection using a deep sequencing approach. The identified microRNAs with specific immune regulating functions will be validated for their target genes. The immune regulating mechanism of microRNA of AEC II against Mycobacteria will be further explored by means of overexpression and silence targeting microRNAs in a AEC II cell host and pathogen model. These studies will provide an insight into the cellular and molecular mechanisms of AEC II in response to Mycobacteria and help us to better understand the immune regulation roles of AEC II in the pathogenesis of tuberculosis.

肺结核是由结核分枝杆菌(Mtb) 感染引起的严重威胁人畜健康的慢性传染病。Mtb感染肺脏时，最先侵染肺泡II型细胞（AEC II）和巨噬细胞，但是目前AEC II细胞的抗结核免疫调控作用尚不清楚；另外，在Mtb感染宿主细胞的免疫应答中microRNA发挥着重要的调控作用，在抗结核免疫机制研究中豚鼠是可靠的动物模型。因此，本项目在豚鼠肺脏免疫信号表达分析与AEC II分离培养的基础上，拟通过对弱毒牛结核分枝杆菌（BCG）感染豚鼠前后AEC II细胞中的microRNA表达谱差异分析，获得免疫调控相关microRNA并进行验证；通过microRNA过表达和沉默等手段在AEC II细胞宿主与病原作用模型上揭示microRNA对结核菌免疫应答中的调控作用，从而在细胞和分子水平上阐明其在结核病发生过程中宿主AEC II细胞的免疫调控机制，进而为抗结核药物/疫苗筛选奠定理论和实践基础。

肺结核是由结核分枝杆菌(Mtb) 感染引起的严重威胁人畜健康的慢性传染病。Mtb感染肺脏时，最先侵染II型肺泡上皮细胞（AEC II）和肺泡巨噬细胞，并且在抗结核免疫机制研究中豚鼠是可靠的动物模型。所以本项目以豚鼠为动物模型，结合人Ⅱ型肺泡上皮细胞系，开展了以下研究内容：①研究了不同胎龄和成体豚鼠肺脏上皮细胞的发育及其不同发育阶段TLRs的表达规律研究；②建立了原代豚鼠Ⅱ型肺泡上皮细胞的消化、分离和体外培养体系；③建立豚鼠肺脏BCG感染模型，并进行microRNA深度测序和miRNA差异表达谱生物信息学分析和验证，初步筛选并验证了部分在抗Mtb感染中免疫调控相关的miRNA，如miR-146a、miR-124、miR-29a、miR-200c等；④针对miR-124靶基因功能及其在Mtb感染AEC II细胞和肺泡巨噬细胞中的调控机理进行了深入研究； ⑤分析了miR-146a在不同毒力Mtb感染Ⅱ型肺脏上皮细胞中表达及其对TLRs信号通路的调控作用；⑥ 初步分析了发育相关Wnt经典信号通路在结核分枝杆菌感染AEC II细胞中的免疫调控功能。通过以上研究内容的完成，证实了miRNA参与了机体抗结核分枝杆菌感染免疫应答中的调控作用，从细胞和分子水平上揭示了其在结核病发生过程中宿主AEC II细胞的免疫调控机制，进而为结核病的发病机制和临床诊断等研究奠定了理论和实践基础。