活动性肺结核血中特异外泌体microRNAs在相关细胞间的穿梭通讯功能及调控机制研究

Tuberculosis remains the leading cause of death worldwide by single pathogen, killing nearly 2 million people each year. China has the world's second largest tuberculosis epidemic. With the occurrence of tuberculosis & HIV coinfection as well as emergence of multi drug-resistant tuberculosis, the epidemic in China is becoming far too serious. However, the existing methods for clinical prevention, diagnosis and treatment of tuberculosis have made so little progress over the past several decades. Further exploration of molecular mechanism involved in tuberculosis may contribute significantly to research and development of new prevention and diagnostic methods as well as new approaches to treatment. Intercellular communication has great importance in all life forms and its disorders are closely related to many diseases. Investigation of intercellular communication is of important significance for understanding of the occurrence, development and therapy of diseases. Recent studies have reported that, as a newly discovered regulation mechanism, exosomal microRNAs-mediated intercellular communication plays a pivotal regulatory role in diverse physiological and pathological processes. MicroRNAs(miRNAs) are small, noncoding RNAs (about 22 nt) that are involved in key biological processes and different diseases by reducing target mRNA stability and/or translation. Recent reports have shown that various types of cells, tissue and diseases have distinctive miRNA expression patterns, which has great theoretic and practical implications in the more detailed interpretation of pathogenesis, development of new diagnosis and treatment methods for diseases. In our previous studies, differential expression of miRNAs was found in serum/sputum/CD4+ T cells from patients with active pulmonary tuberculosis compared to healthy controls. The data indicate that differential expression of miRNAs is involved in occurrence of active pulmonary tuberculosis. However, up to now, little is known about function and regulation mechanism of exosomal miRNAs-mediated intercellular communication in the tuberculosis. Based on our previous research, aim of the project is to further identify specific exosomal miRNAs in blood related to active pulmonary tuberculosis, explore their function during intercellular communication and regulation mechanism using miRNA chip, real time quantitative PCR, bioinformatics analysis, cell co-culture system, animal model of tuberculosis, and methods for construction of expression vector. This work will elucidate new molecular mechanism for the occurrence of tuberculosis from the novel way of exosomal miRNA mediated intercellular communication, which has important theoretic and practical significance for the diagnosis, prevention and control of tuberculosis.

结核病是当前由单一致病菌导致死亡最多的传染病。深入研究结核病发生的分子机制及寻找新的防治策略是亟待解决的科学问题。细胞间通讯异常与疾病的发生发展密切相关。新近研究发现：外泌体穿梭miRNA通过胞间通讯方式介导靶基因沉默参与了很多疾病过程。我们前期系列研究表明：活动性肺结核体液、免疫细胞中多个miRNA的表达发生了显著改变，且它们调控的许多靶基因与结核病程密切相关，提示结核发病机制与miRNA表达异常有关。本项目拟应用miRNA芯片、定量PCR、细胞共培养、结核模型构建、生物信息学分析等方法进一步鉴定活动性肺结核血中特异的外泌体穿梭通讯miRNA；在体外、体内探索它们通过胞间通讯介导巨噬细胞、T细胞、树突状细胞等相关细胞发生功能异常中的作用；确认穿梭miRNA调控靶基因及信号通路对结核感染的影响。在miRNA胞间通讯调控水平探索和揭示结核病发生发展的分子机制，并为其防治提供理论和实验依据。

项目背景.深入研究结核病发生的分子机制及寻找新的防治策略是亟待解决的科学问题。细胞间通讯异常与疾病的发生发展密切相关。新近研究发现：外泌体穿梭miRNA通过胞间通讯方式介导靶基因沉默参与了很多疾病过程。我们前期系列研究表明：活动性肺结核体液、免疫细胞中多个miRNA的表达发生了显著改变，且它们调控的许多靶基因与结核病程密切相关，提示结核发病机制与miRNA表达异常有关。.主要研究内容.收集合格的活动性肺结核患者临床血样本及对照组样本；分离纯化及鉴定外周血外泌体；应用miRNA芯片、定量PCR、细胞结核模型构建、生物信息学分析等方法进一步鉴定活动性肺结核血中差异表达的外泌体miRNA；研究调控差异miRNA表达对巨噬细胞内结核分枝杆菌（Mycobacterium tuberculosis, Mtb）的影响，在miRNA胞间通讯调控水平探索和揭示结核病发生发展的分子机制，并为其防治提供理论和实验依据。.重要结果与关键数据.完成了外周血外泌体的分离纯化及鉴定；与对照组相比，发现结核患者血外泌体中有6个差异miRNA；生物信息学分析发现差异miRNA预测的调控靶基因分子功能主要为蛋白结合、离子结合、核苷酸结合等功能，主要参与MAPK信号通路、钙离子信号通路等；发现结核患者外周血外泌体中有81个差异蛋白质；完成了差异miRNA对胞内Mtb影响的研究并对差异miRNA的靶基因进行了预测及功能分析；完成了结核患者外周血脂代谢改变的分析，发现脂代谢可作为肺结核诊断及治疗的敏感指标；完成了Mtb毒力基因MT3876、Rv0867c、TrpD编码蛋白的生物学功能预测分析等。.科学意义.本项目从一个全新的角度—外泌体miRNA的细胞间通讯信号转导的新途径，阐明结核病发生发展过程中的分子机制。研究结果可为后续外泌体miRNA、蛋白、脂质在胞间通讯调控水平探索诊断结核的方法学突破与寻求结核病治疗的新途径提供科学的理论依据与实验依据；同时也为探索影响其它肺部疾病进程的外泌体分子机制等问题，开创了一个全新思路与有益探索。