P2Y4受体信号通路调控雪旺氏细胞迁移对喉返神经再生的作用

Schwann cells (SCs) play a pivotal role in the process of peripheral nerve regeneration. Upon injury, de-differentiated SCs begin to proliferate, differentiate, secrete growth factors and migrate to the lesion site engaging to constitute the nerve bridge. SCs provide the regenerated axons with guidance signals and growth supporting, but also multispirally wrapped up the axons to remyelination. Emerging evidences indicated that migratory ability of Schwann cells contributed to the outcomes of nerve regeneration. However, the signaling mechanism regulating Schwann cell migration is very complicated. As a specific kind of neurotransmitter, purins widely enganged in the functional regulation of nervous systems via bounding different purinergic receptors. Our previous study has demonstrated directional collective cell migration of P2Y4-positive SCs out of the nerve stumps to guide regrowing neurofilament-positive axons across the wound. And the in vitro experiment also implicated that agonists or antagnists of P2Y4 receptor can promote or inhibit the migration of cultured rat primary SCs. Based on the data, we hypothesize that P2Y4 signaling may mediate migration of SCs to promote laryngeal nerve regeneration. However, the mechanism of P2Y4 receptor signaling transduction regulating SCs remains to be elucidated. For this purpose, gene knock-down and specific inhibitor in cultured Schwann cells are used to verify the definite effect of P2Y4 receptor. And the pivotal cascades of P2Y4 signaling are validated by constituted activation and inactivation of migration-related signaling components. Finally, the effect of P2Y4 receptor on SCs is demonstrated in vivo using a rabbit model of recurrent laryngeal nerve injury and regeneration. The present study aims at exploring signaling transduction mechanism and the role of P2Y4 receptor in the regeneration process of recurrent laryngeal nerve, which is expected to highlight the regulatory effect of P2Y4 receptor in SCs, and to provide a novel and potential therapeutic strategy for the treatment of peripheral nerve injury.

喉返神经修复后神经再生往往不完全，影响喉功能的恢复。雪旺氏细胞定向迁移能力是影响神经再生程度的重要因素。嘌呤能受体P2Y家族成员可激活多种信号通路调控神经再生，申请人证实喉返神经再生过程中，强烈表达P2Y4受体的雪旺氏细胞向损伤处定向迁移，形成雪旺氏细胞带包饶再生轴突形成髓鞘，而P2Y4受体拮抗剂可延缓神经再生速率和程度；激活P2Y4受体明显促进雪旺氏细胞迁移，提示P2Y4受体可能通过调控雪旺氏细胞迁移诱导轴突定向生长而促进喉返神经再生，但机制尚不清楚。本课题采用雪旺氏细胞/神经元共培养体系研究雪旺氏细胞诱导轴突定向生长的作用；通过组成性失活/激活技术探讨P2Y4受体调控雪旺氏细胞迁移的信号机制；利用喉返神经再生动物模型，明确P2Y4受体对喉返神经再生的作用。从分子、细胞和组织水平，阐明P2Y4受体调控雪旺氏细胞迁移的信号机制及对喉返神经再生的作用，旨在为促进喉返神经再生寻找新的治疗靶点。

周围神经损伤常引起相应器官功能缺陷，神经再生后也难以完全恢复正常。雪旺氏细胞（SC）定向迁移能力是影响神经再生的重要因素，其在神经损伤后去分化并大量增殖、迁移和再分化，诱导再生轴突定向生长并最终形成髓鞘。嘌呤能受体P2Y家族可激活多种信号通路调控神经再生。研究发现P2Y4受体参与周围神经损伤后的病理生理改变及再生过程。申请人也证实SC上P2Y4受体在喉返神经再生过程中起重要作用，但机制尚不清楚。. 本课题拟利用周围神经再生动物模型及雪旺氏细胞-神经元共培养体系，明确P2Y4受体对神经再生的作用，阐明其调控SC迁移、轴突诱向再生的分子及信号转导机制。. 我们通过选用Nuerolac可吸收共聚高分子外周神经套接管构建大鼠面神经切断损伤再生室，采用ALZET植入式胶囊渗透压泵行药物干预，建立稳定模型。术后6周通过大体观察各组面肌运动恢复情况、检测面肌诱发电位以及再生神经及肌肉的形态学研究，发现激活P2Y4 受体有髓神经纤维数和平均直径及髓鞘厚度、面肌纤维数目和直径明细改善，明确促进了轴突再生和面肌功能的恢复。通过双重免疫荧光标记及Peggy Sue微量蛋白定量分析、RT-PCR等分子生物学研究方法，发现ERK1/2 、PI3K、p38MAPK和RhoA这四个通路在面神经损伤后被激活，且表达具有各自的时间效应，提示其参与神经再生的调节且发挥协调效应。激活P2Y4 受体后上述信号分子的表达明细上调，同时通过调控再生早期NCAM、Vimentin等表达促进了SC的迁移和轴突的延伸以及神经再生（髓鞘碱性蛋白MBP的表达明显上调）。. 本研究通过阐明P2Y4受体调控雪旺氏细胞迁移的信号机制及对神经再生的作用，旨在为促进周围神经再生寻找新的治疗靶点。为寻找促进面肌功能恢复的新的药物治疗作用靶点奠定了基础，也为促进面神经等周围神经再生提供了新的思路和理论依据。