巨噬细胞源性外泌体microRNA在紫杉醇诱发的痛性周围神经病变中的作用及机制研究

The peripheral painful neuropathy induced by paclitaxel, a widly used chemotherapeutic drug, prohibits the application of this medicine in patients. We previously reported paclitaxel induced the infiltration of macrophage in DRG and drave apoptosis of the neurons. Further more, we found macrophage was indispensable in the neuron apoptosis by cell co-culture study. However, the intercellular communication between the macrophage and DRG neuron is still unclear. Recently, exosome was confirmed as an key mediator of intercellular communication. MicroRNA is one of the most important component of exosome, which plays a critical role in pain regulation. Further more, we extraced the macrophage-derived exosome and treated the neurons with paclitaxel. Remarkble apoptosis were ob Taken together, we supporse that macrophage-derived exosome microRNA probably observed in neuron following the incubation. We sequenced the macrophage-derived exosome microRNA, 50 differentialy expressed microRNA was detected. According to this, we hyposed that macrophage-derived exosome microRNA is an important intercellular communication mediator in paclitaxel induced peripheral painful neuropathy. On these basis, we plan to identify the key macrophage-derived exosome microRNA in paclitaxel induced peripheral painful neuropathy by using in vitro and in vivo model, patch clamp; reveal the target gene and signaling of these exosome microRNA via transfection, transefection and 3' UTR-Luciferase ect.; evaluate the clinical significance of these molecue through the clinical resource. This project will provide the novel target of paclitaxel induced peripheral painful neuropathy.

紫杉醇诱发的痛性周围神经病变（CIPPN）限制其临床应用。我们前期报道紫杉醇诱导巨噬细胞在DRG浸润，导致神经元凋亡；细胞共培养发现该过程中神经元凋亡必需巨噬细胞参与，但两者间信息通讯尚未阐明。最新发现外泌体是细胞间信息通讯重要途径，miRNA是外泌体中关键分子，在疼痛调控中举足轻重。我们进一步提取巨噬细胞外泌体与紫杉醇共处理神经元细胞，同样可诱导神经元凋亡；测序巨噬细胞外泌体miRNA，发现50种差异表达miRNA。我们假设：巨噬细胞源外泌体miRNA可能介导与DRG神经元信息通讯，在紫杉醇诱发的CIPPN中发挥重要作用。本项目拟在前期基础上通过体内外模型、电生理等，鉴定紫杉醇诱发CIPPN中关键巨噬细胞源外泌体miRNA；利用转染、3' UTR-Luciferase等，明确miRNA靶基因及信号通路；结合临床，评估上述miRNA临床意义。本项目将为紫杉醇诱发的CIPPN提供新的分子靶标

紫杉醇是广泛用于治疗癌症的化疗药，其可诱发痛性周围神经病变（CIPPN）且病理机制复杂，目前尚无有效的治疗方法。本项目拟研究外泌体microRNA介导巨噬细胞和神经元间通讯及其在紫杉醇诱导神经凋亡中的作用，探讨其在外周敏化的作用，可为治疗紫杉醇诱发的神经病理性疼痛提供新的潜在分子靶标。主要研究内容：（1）巨噬细胞外泌体与DRG神经元之间的细胞通讯在紫杉醇诱发的CIPPN中的作用;（2）筛查并鉴定在紫杉醇诱发的CIPPN中发挥重要作用的巨噬细胞源性外泌体microRNA;（3）巨噬细胞源关键外泌体microRNA在紫杉醇诱发CIPPN中的功能学研究（4）巨噬细胞源关键外泌体microRNA在紫杉醇诱发的CIPPN中的调控机制。实验结果表明：（1）紫衫醇刺激巨噬细胞后，其分泌的外泌体粒径和表达增加;（2）巨噬细胞分泌的外泌体可被DRG神经元摄取，并诱导机械痛敏和热痛敏；（3）已经筛选出了紫衫醇处理巨噬细胞后其分泌的外泌体microRNA（microRNA-141-3p）；（4）炎症是神经病理性疼痛的关键环节，而巨噬细胞源外泌体内的microRNA-141-3p负性调控神经元IκB-α，提示紫衫醇处理巨噬细胞后其分泌的外泌体microRNA-141-3p可能通过调控神经元的炎症介导CIPPN。本课题科学意义：(1)巨噬细胞源外泌体可能是治疗紫杉醇诱发的CIPPN的新的潜在靶标；（2）紫杉醇化疗后患者血清中外泌体关键microRNA可为诊断其神经病理性疼痛的潜在生物标记物。