脉络丛上皮细胞源性外泌体miR-146a介导外周炎症信号血-脑通讯在术后谵妄中的作用及机制研究

Postoperative delirium（POD）, an acute, transient, fluctuating disturbance in attention, cognition, and level of consciousness, is a common postoperative complication. It is associated with increased morbidity, mortality and cost. However, at the present time, POD remains a wholly clinical diagnosis; its classic animal models, neuropathogenesis and targeted intervention(s) remain largely to be determined. Recent studies have suggested the important roles of choroid plexus epithelium cells (CPECs) derived exosomal microRNAs(miRNAs) on the blood-brain communication during peripheral inflammation. In our previous studies, we established a potential animal model using “CAM in Mice” battery to study POD in mice and found that microRNA-146a(miR-146a) contributes to the neuroinflammation in hippocampus of POD mice; in addition, the amount of exosomes and exosomal miR-146a expression increased in cerebrospinal fluid (CSF) isolated from patients with POD and POD mice; moreover, exosomes containing miR-146a (Exo-miR-146a) in the CSF might originate from CPECs. Therefore, we hypothesized that CPECs derived exosomal miR-146a regulates blood-brain communication of systemic inflammation during POD. The present project aims to investigate the roles of CPECs derived exosomal miR-146a on mediating inflammatory signaling from blood to brain, to assess the rescue effects of up-regulated expression of Exo-miR-146a on neuroinflammation induced by POD, and to evaluate the diagnostic potential of CSF Exo-miR-146a as a POD biomarker. We will test our hypothesis at the live animal level, cultured cell level, and cultured choroid plexus explant level. Behavior test, high-throughput sequencing technology, bioinformatics analysis, histology/ immunohistochemistry, electrophysiology and molecular biology approaches will be used for the proposed studies. A better understanding of the roles of CPECs derived exosomal miR-146a on POD neuropathogenesis will help to identify new mechanisms of POD and reveal potential new diagnostic and therapeutic approaches for POD.

术后谵妄（POD）是麻醉学与神经科学研究热点，其具体机制不清。研究表明，脉络丛上皮细胞（CPECs）分泌的外泌体microRNAs可介导外周炎症信号的血-脑通讯。课题组已证实，microRNA-146a（miR-146a）调控POD小鼠海马炎症反应；伴随外周血IL-6表达水平增高，POD患者和小鼠脑脊液中外泌体数量及外泌体miR-146a表达水平增加，且CPECs可能是外泌体miR-146a的来源细胞。由此推测CPECs分泌的外泌体miR-146a可能介导POD外周炎症信号向脑的传递。本项目从整体动物，培养细胞及脉络丛外植体三个层面，观察CPECs源外泌体miR-146a在POD外周炎症信号血-脑通讯中的作用；同时观察上调外泌体miR-146a表达对POD炎症反应的干预作用；并研究脑脊液外泌体miR-146a作为POD诊断标志物的可行性，为阐明POD发病机制及诊断和防治提供新的靶点及工具。

术后谵妄（postoperative delirium, POD）是老年患者麻醉手术后常见的中枢神经系统并发症，其发病机制不清。课题组研究发现MicroRNA-146a可通过调节海马炎症反应调控POD的发生发展。此领域既往研究多聚焦于血脑屏障受损，课题组靶定血脑脊液屏障（Blood-Cerebrospinal Fluid Barrier ，BCSFB)，从新的方向探索了POD外周炎症信号向中枢传递的机制。研究结果提示POD大鼠BCSFB结构受损，通透性增加，腺苷A2a受体可通过调节RhoA/ROCK2信号通路调控BCSFB通透性，调控POD症状，提示腺苷A2a受体可能是POD治疗靶点。在上述研究基础上，基于BCSFB和谵妄的密切关联与目前缺乏理想BCSFB体外模型的研究现状，课题组依据人体脉络丛解剖结构与生长环境特点，采用人源性脉络丛上皮细胞和微血管内皮细胞共培养，结合微流控技术，设计和构建了高度仿生、便于观察和取材的 BCSFB体外模型（BCSFB-on-chip）。进一步的表征和功能鉴定结果显示，工程化BCSFB-on-chip明显促进TTR，CD31, Glut-1和AQP-1等BCSFB组织特异性标志物及紧密连接蛋白ZO-1的表达，分子量依赖性地降低屏障通透性。RNA-seq结果显示，差异性基因表达富集至血管生成，细胞连接，膜装配及物质转运等与BCSFB生理功能密切相关的通路。课题组还在神经炎症环境中验证了上述表征和功能变化。这些结果均提示BCSFB-on-chip可成功模拟生理及病理条件下血脑脊液屏障结构，可为POD机制研究及中枢药物递送系统研发提供有力工具。. 此外，课题组开展了POD新型分子标志物研发的一项多阶段临床研究。该研究选取老年膝髋关节置换术后谵妄患者的外周血中差异性表达的脑源性外泌体lncRNAs作为候选分子标志物，结合临床病例对照研究，观察标志物与POD的发生率、严重程度，亚型及病程进展的关系，同时验证其预测POD的敏感性和特异性，并观察其稳定性，以期为POD预测提供新工具。目前，已经完成大部分病例数据及样本收集，外泌体分离，外泌体RNA提取，微量RNA建库和测序等内容。. 综上所述，本研究为POD机制研究及治疗提供新的靶点和工具，同时为高危患者筛选提供新的生物标志物，有助于从多维度解决POD这一围术期医学难点问题。