喉返神经轴突再生程度不足是声带麻痹患者喉功能恢复不佳的重要原因。新近发现的神经突起因子Neuritin是外在因素共同作用的关键下游靶点，我们的前期研究及文献报道其在神经轴突再生中起着重要作用，但关于其上游调控机制尚不太清楚。申请人证实喉返神经再生过程中嘌呤能受体P2Y1和Neuritin表达上调，而抑制Neuritin可显著阻断P2Y1促轴突生长效应。P2Y1受体可激活ERK、p38、PI-3K等信号通路，后者与CREB转录激活密切相关。由此推测：P2Y1受体可能通过ERK、p38或PI-3K调控CREB/Neuritin通路，发挥促进神经轴突再生的作用。本课题拟通过在体和离体实验，采用组成型激活/失活技术，从分子、细胞、组织和在体水平，阐明P2Y1调控CREB/Neuritin的信号转导机制及对轴突再生的作用，旨在为促进喉返神经再生和喉功能恢复，寻找新的治疗靶点，提供理论及实验依据。

Vocal fold paralysis (VFP) caused by the injury to recurrent laryngeal nerve (RLN) is a common disease in otorhinolaryngology department, which severely impairs the quality of life for those patients. Our previous investigations revealed that a varying degree of spontaneous axons regeneration occurred in symptomatic VFP patients who exhibited typical denervation potential and poor regenerative potential in laryngeal electromyography examination. These histologic and electrophysiologic evidences indicated that inadequately regenerative degree of RLN axons resulted in the unsatisfactory recovery of laryngeal function in those symptomatic VFP patients, and after undergoing laryngeal reinnervation as well. Hithreto, the mechanism of regulating axonal regeneration remains unclear. Recent study focused on Neuritin, coded by candidate plasticity-related gene 15 (CPG15), which plays an important role in the central and peripheral nervous systems. It was reported that both NMDA/CaMKs and BDNF/TrkB signaling pathways can modulate the transcriptional activation of CREB (cAMP-response element binding protein), then CREB up-regulates the expression level of Neuritin via binding to the promoter region of Neuritin. Our in-vivo experiment found that purinergic receptor P2Y1 and Neuritin up-regulated in the regenerating RLNs. Furthermore, the positive effect of P2Y1 promoting axonal regeneration was almost abolished by the neutralized antibody against Neuritin in the cultured primary motoneurons. As a member of G-protein coupled protein family, P2Y1 receptor exerts an extensive and important effect in the physiopathological process through modulating the signaling molecules of PKA, ERK/MAPK, p38/MAPK, PI-3K/AKT and so on. These signaling pathways mentioned above have proven to be closely correlated to the transcriptional activation of CREB. Whether P2Y1 receptor regulates the Neuritin expression via those signaling pathways is unknown yet. Together, we hypothesized that P2Y1 receptor may modulate the transcriptional activation of CREB to regulate the Neuritin via PKA or ERK/MAPK or p38/MAPK or PI-3K/AKT signaling pathway. For this purpose, we will establish a mice model of RLN regeneration and the cultured primary motoneurons. In vivo and in vitro experiments, using some specific drugs and gene interference, are designed to demonstrate the spatio-temporal expression and localization of P2Y1, CREB and Neuritin in the process of RLN regeneration, and elucidate the signaling transduction mechanisms of P2Y1 regulating CREB /Neuritin. This study is expected to explore a potentially therapeutic target to promote axonal regeneration of RLN. The satisfactory rehabilitation of laryngeal function may be obtained after laryngeal reinnervation by this novel therapeutic strategy.