肺泡上皮细胞外泌体介导非编码RNA对巨噬细胞抗结核分枝杆菌感染TLR信号的调控作用机制

Pulmonary tuberculosis is a chronic fatal zoonotic infectious disease caused by an infection of Mycobacterium, which has significantly hampered a normal and stable development of animal production industry. Both alveolar epithelial cells and alveolar macrophages are the targets and immune cells of mycobacterial infections which have been demonstrated to play key roles in maintenances of the immunological homeostasis during an infection,but the regulatory role of interaction between these two cells remains unclear.In recent years, studies have shown that exosome played an important role in regulation in a wide variety of diseases developing process，mainly through the realease of miRNA and LncRNA containing factors and genetic materials with various biological activities. And its role in prevention and control of TB has not been reported..Based on the research background, the goal of present proposal aims to identify miRNA and LncRNA in exosome of epithelial cells infection with Mtb related to infection of macrophage anti Mycobacterium tuberculosis through High throughput sequencing (RNA-Seq)technology. Over expression vector/mimic and siRNA vector/inhibitor will be constructed to transfect U937 cells respectively,and then the transfected macrophage will be infected with Mtb in order to clarify regulatory role of the specific noncoding RNA molecules of the alveolar epithelial cells in Macrophages infection with Mtb. The research results will provide further insight into the pathogenesis of tuberculosis (TB).

结核病是由结核分枝杆菌引起的慢性致死性人兽共患病，严重影响畜牧业健康稳定发展。肺泡上皮细胞和巨噬细胞都是结核分枝杆菌的靶细胞和免疫细胞，在抗结核分枝杆菌感染过程中对维系机体免疫反应的动态平衡发挥关键作用。但两种细胞之间相互作用的调控机制尚未见相关报道。近年研究表明，外泌体主要通过释放含miRNAs和LncRNA等生物活性物质在多种疾病的发生发展过程中发挥了重要的调控作用，而其在结核病防控中的作用还未见报道。.基于以上研究背景，本项目旨在通过高通量测序（RNA-Seq）技术筛选结核分枝杆菌感染肺泡上皮细胞后,外泌体中与巨噬细胞抗结核分枝杆菌感染有关的LncRNA和miRNA，通过构建其过表达载体/mimic和干扰载体/inhibitor并转染U937细胞后并用结核分枝杆菌感染,以期阐明上皮细胞中特定非编码分子在巨噬细胞抗结核杆菌感染中的调控作用,对深入理解结核病发病机制具有重要的科学意义。

研究背景：结核病是由结核分枝杆菌引起的慢性致死性人兽共患病，严重影响畜牧业健康稳定发展。肺泡上皮细胞和巨噬细胞都是结核分枝杆菌的靶细胞和免疫细胞，在抗结核分枝杆菌感染过程中对维系机体免疫反应的动态平衡发挥关键作用。但两种细胞之间相互作用的调控机制尚未见相关报道。肺脏纤维化是肺脏组织长期损伤修复异常而导致的致死性的慢性疾病，也是各种间质性肺疾病的最终病理表现。上皮细胞间质转化是上皮细胞转化为具有间质表型细胞的生物学过程，是多种纤维化疾病发生发展过程中的关键病程。外泌体作为一种包含多种生物大分子的囊泡，在细胞间通讯中发挥重要作用。.主要研究内容：.1..肺脏上皮细胞外泌体对巨噬细胞感染 BCG诱导的免疫调控作用研究.2..脂多糖刺激巨噬细胞来源外泌体对TGF-β1诱导肺脏上皮细胞间质转化的影响.重要结果及关键数据：.1..BCG感染和未感染上皮细胞来源的外泌体可上调炎症因子及炎症相关蛋白的表达，促进炎症反应。BCG感染后，正常上皮细胞来源的外泌体可显著增强巨噬细胞的凋亡和自噬，BCG感染来源的外泌体与正常的外泌体相比较，巨噬细胞的凋亡与自噬水平较弱。推测当BCG感染巨噬细胞后，正常上皮细胞来源的外泌体可通过调控细胞的凋亡和自噬来促进巨噬细胞清除入侵的结核分枝杆菌，而BCG感染上皮细胞来源的外泌体不利于巨噬细胞对结核分枝杆菌的清除。其中外泌体对BCG诱导的巨噬细胞自噬的调控作用依赖于mTOR信号通路。.2..脂多糖刺激巨噬细胞来源外泌体参与上皮细胞间质转化。它通过上调p-Smad2/3的表达水平，激活TGF-β1/Smad2/3信号转导通路，造成下游转录因子的高表达，进而促进上皮细胞间质转化。.科学意义：为上皮细胞/巨噬细胞相互作用在抗结核分枝杆菌感染的免疫调控研究提供新思路；了解脂多糖刺激巨噬细胞来源外泌体与上皮间质转化有关的调控机制可为进一步研究肺纤维化发病机制及肺纤维化防治提供新思路。