梅毒螺旋体诱导的巨噬细胞源性外泌体在血管内皮损伤中的作用及机制研究

Syphilis is the major sexually transmitted infection caused by the bacterial spirochetal Treponema pallidum subsp. pallidum (T. pallidum，Tp). It has been argued that the inflammatory and immune responses mediated primarily by Tp may play an important role in vascular dysfunction. However, the mechanism by which Tp activated lymphocytes and promoted endothelial inflammation have not been identified. Our previous studies showed that exosomes were secreted by macrophages after treatment with Tp. We also found that these exosomes contained substantial amounts of microRNA and Tp membrane protein Tpp47. These exosomes can up-regulate expression of VCAM-1 in endothelial cells. In this study, we will induce macrophages with Tp and harvest exosomes secreted by macrophages. The effects of exosomes on dysfunction of the endothelial cells were identified by real-time PCR, Western blot and other methods. To address the role of Tpp47 protein in activation and permeability of endothelial cells, lentivirus transfection, cell-ELISA and other methods are used. The mechanism of migratory endothelial cells regulated by Ago2-miR-210-3p RISC will be further investigated. Dual Luciferase Reporter Gene Assay is used to identify VEGF gene as a target gene of miR-210-3p. Finally, we will study the modulation of miR-483-5p gene on dysfunction of the endothelial cells via targeting eNOS gene. The results of our study will be useful for identifying the role of macrophage-derived exosomes in the pathogenesis of T. pallidum, and may be a potential target for clinical management of syphilis.

梅毒是由梅毒螺旋体( T. pallidum，Tp)感染所致的严重危害人类健康的性传播疾病。目前Tp致病机制尚未阐明，研究认为Tp诱导的炎症反应是导致梅毒血管内皮损伤的主要因素，但具体致炎机制还不清楚。本课题组前期研究发现Tp可诱导巨噬细胞释放外泌体，其携带特异性miRNA和Tpp47膜蛋白，并可活化血管内皮细胞。本课题拟分离Tp诱导的巨噬细胞源性外泌体，明确其对血管内皮细胞功能影响；其次采用慢病毒包装及转染等技术，研究外泌体中Tpp47在细胞内活化内皮细胞及调控细胞单层通透性机制；同时采用双荧光素酶报告基因实验等方法验证外泌体中Ago2-miR-210-3p RISC通过调控VEGF促进血管内皮细胞迁移的机制；最后，采用RNA免疫共沉淀等技术，研究miR-483-5p对eNOS的调控及导致血管内皮损伤机制。本课题为阐明巨噬细胞源性外泌体在梅毒血管内皮损伤中的作用及作为干预靶点提供理论基础

梅毒是由梅毒螺旋体( T. pallidum，Tp)感染所致的严重危害人类健康的性传播疾病。梅毒发病机制尚未阐明，研究认为Tp诱导的炎症反应是导致梅毒血管内皮损伤和造成心血管不良结局的主要因素，但具体作用机制尚不清楚。针对本课题组前期研究发现，我们首先开展Tp诱导巨噬细胞源性外泌体对血管内皮细胞功能影响的研究，发现其可以提高脐静脉内皮细胞（HUVEC）对THP-1细胞的黏附能力，增强HUVEC单层细胞通透性；进而对外泌体中miRNA的作用进行研究，发现外泌体中miR-146a-5p可通过抑制血管内皮细胞的靶基因JAM-C使血管内皮细胞通透性降低及单个核细胞跨内皮迁移降低，在炎症反应致血管内皮损伤中发挥重要作用。另外，我们对课题实施中发现的其他有价值的结果开展了研究，发现：（1）血浆中miR-223-3p可以靶向抑制NLRP3炎症小体的活化并介导内皮细胞的焦亡，表明其可能是梅毒患者的一个潜在治疗靶点；（2）Tp92蛋白可能在通过诱导趋化素（chemerin）的分泌介导内皮细胞活化过程中起重要作用；（3）Tp可改变人脑微血管内皮细胞（HBMECs）的基因表达谱，为进一步研究新的神经梅毒发病机制提供了依据；（4）我们鉴定出一个新型tp0548基因型Qn，拓展了tp0548基因数据库，表明了tp0548基因序列的遗传多样性。本课题研究主要集中于巨噬细胞源性外泌体及外泌体中miRNA、血浆中miRNA、Tp膜蛋白在血管内皮损伤中发挥的作用及机制，研究结果为了解Tp损伤血管内皮的作用及机制提供了新的思路和依据，对防治梅毒及其不良结局具有重要意义。