不同毒力MTB菌株与宿主巨噬细胞互作过程中的非编码RNA免疫调节机制网络研究

According to WHO tuberculosis (TB) reports, TB has exceeded HIV as the leading lethal infectious disease globally for three consecutive years. Due to the spread of drug-resistant tuberculosis, the global situation of tuberculosis control is austere. Macrophage is the major defense against Mycobacterium tuberculosis (MTB). As a facultative intracellular bacterium, in particular the highly virulent strains, MTB nevertheless have evolved a variety of evasion strategy against host macrophage immune system. The course of tuberculosis was largely determined by the interactions between MTB and macrophages. In this process, non-coding RNAs may play an important role. Comprehensive analysis of non-coding RNA immune regulation network is very important for thoroughly investigating the exquisite regulation mechanism of the immune system against MTB and the disease development. Through high throughput sequencing technology and other related technologies, this project aims to screen the panoramic expression profiling of the lncRNAs and miRNAs, construct the immune regulatory network of non-coding RNAs, and further explore their internal regulation related to "MTB infection" and "MTB virulence". Finally, we will excavate and experimentally verify the functions of potential non-coding RNAs which may play key roles in the regulation network, and thus provide powerful theoretical support and a new resolution for clinical diagnosis, prevention and treatment on tuberculosis.

根据WHO结核病报告，结核病（TB）已经连续3年超过艾滋病成为全球第一传染病杀手，并且由于耐药结核流行等原因，使其全球防控形势更加严峻。巨噬细胞是抵御结核分枝杆菌（MTB）感染的主要屏障，但作为兼性胞内菌的MTB，已进化出多种对抗巨噬细胞的免疫逃逸策略。巨噬细胞与MTB的相互作用结果很大程度上决定了结核病的病程转归，而非编码RNA则可能在其中发挥重要功能。全面解析非编码RNA免疫调节机制网络，无疑对于深入揭示抗结核免疫的精细调控机制以及结核病病程发展规律具有极其重要的意义。本项目拟借助高通量测序技术，绘制感染MTB的巨噬细胞中lncRNA和miRNA的全景式表达图谱，构建非编码RNA免疫调控网络，并探究它们与“MTB感染”和“MTB菌株毒力”的内在调控规律。我们还将进一步借助实验对可能发挥关键作用的非编码RNA进行功能解析，为结核病的临床诊断、预防及治疗研究提供理论基础及新思路。

尽管结核病的全球防控形势日益严峻，但是研究者们仍然缺乏对结核分枝杆菌感染宿主巨噬细胞机制的全面了解。作为抵御细菌感染的主要屏障，巨噬细胞与病原菌的相互作用结果很大程度上决定了疾病的病程转归。本项目通过构建不同毒力结核分枝杆菌感染巨噬细胞的模型，完成转录组学测序分析，展示了巨噬细胞和外泌体中基因的全景式动态表达谱，并完成了感染模型中功能通路的预测和分析，提出低毒力和高毒力细菌感染造成巨噬细胞不同免疫应答的潜在调控机制。本项目获得的主要研究结果为：1）成功构建低毒力H37Ra和高毒力H37Rv的结核分枝杆菌感染宿主巨噬细胞的模型，并完成了感染模型的表型鉴定和相关分析；2）分离获得了高纯度巨噬细胞外泌体，为后续转录组学测序奠定基础；3）完成巨噬细胞和外泌体全部RNA的提取和测序，获得巨噬细胞和外泌体中mRNA的全景式表达图谱；4）发现不同毒力Mtb感染引起不同程度免疫应答，具体表现为低毒力Mtb更容易被巨噬细胞识别，并激活更强大的分子免疫网络以清除致病菌，而高毒力Mtb感染通常伴随免疫逃逸，并显著活化细胞凋亡和自噬相关功能分子网络；5）提出拟干扰素通路假说，尽管不表达干扰素基因，但是巨噬细胞中活化的通路网络能够模拟干扰素功能杀菌；6）我们还利用了粒细胞模型验证了拟干扰素通路的假说，并在该模型中发现长非编码RNA的广泛表达，不仅具有细胞类群表达特异性，还在粒细胞成熟和免疫功能中发挥潜在的转录调控机制。我们的研究结果为结核病的临床诊断、预防及治疗研究提供理论基础及新思路，也为天然免疫系统的抗菌机制提供了关键的数据支持和新的研究方向。