脊髓损伤（SCI）治疗主要包括神经保护与神经修复两方面，其中神经干细胞（NSCs）是神经修复潜在治疗方法之一。课题组在明确火针疗法治疗SCI临床疗效，并通过相关实验确定该法对SCI具有神经保护作用及促NSCs增殖分化趋势的基础上，本研究拟通过复制SCI大鼠整体模型，利用血清和脑脊液药理学建立离体模型，观察火针疗法干预后内源性及离体NSCs增殖与分化相关蛋白表达，并进一步观察NSCs向神经元分化中Wnt/β-Catenin与ERK1/2多信号通路上关键靶点的活化表达。在此基础上，通过细胞培养，采用siRNA等技术，检测GSK3β表达变化，以明析该基因为调控Wnt/ERK多信号途径中的串话关键点，以期从整体到细胞多层次探讨火针疗法通过Wnt/ERK多信号通路及相互间协同调控内源性NSCs向神经元分化的靶向途径，探索该法对SCI后脊髓神经修复效应与机制，为火针治疗中枢神经损伤性疾病提供实验依据。

The treatment methods of spinal cord injury (SCI) include neuroprotection and neural restoration, and neural stem cells (NSCs) is one of the potential methods of neural restoration. Based on the specifically clinical effect of fire needle therapy in SCI and the evidence that fire needle therapy has certain neuroprotective effect and could promote the proliferation and differentiation of NSCs in SCI through correlative animal experiment, we duplicate the intergral rat model of SCI and establish in vitro model using of serum pharmacology and cerebrospinal fluid pharmacology, to observe the expression of proliferation and differentiation-associated proteins of endogenous and in vitro NSCs after intervened by fire needle therapy, and to further observe the active expression of key targets of Wnt/β-catenin and ERK1/2 multiple signal pathway during neuronal differentiation of NSCs. Based on that, through cell culture, with the method of siRNA,testing the expression of GSK3β,to make sure if gene GSK3β is the crosstalk key target in the method to regulate Wnt/β-catenin and ERK1/2 multiple signal pathway, to explore the target way to regulate the neuronal differentiation of NSCs of fire needle therapy through Wnt/ ERK multiple signal pathway and intercoordination in many levels from entirety to cell, to explore the effect and mechanism of neural restoration after SCI of fire needle therapy, and to provide experimental references of central nervous system injury diseases treated by fire needle therapy.

脊髓损伤是一种严重的中枢神经系统损伤性疾病，导致患者瘫痪、感觉缺失及自主神经障碍，给个人、家庭及社会造成的沉重的负担与痛苦。目前，脊髓损伤的治疗主要集中于两方面，一是神经保护，主要通过减轻或消除继发性病理反应（细胞凋亡）而达到保护残存轴突和神经元细胞的作用；其二是神经再生与修复，即促进神经组织——主要是神经元的再生与恢复，再建神经元突触间的联系。其中，神经干细胞治疗被认为是目前治疗脊髓损伤最具潜力的方法之一。本研究通过复制脊髓损伤大鼠模型，利用血清药理学建立离体模型，观察火针疗法干预后内源性及离体神经干细胞增殖与分化相关蛋白表达，并进一步观察神经干细胞向神经元分化中 Wnt/β-Catenin 与 ERK1/2 多信号通路上关键靶点的活化表达。在此基础上，通过细胞培养，采用siRNA 等技术，检测 GSK3β沉默前后相关蛋白及基因表达变化，以明析该基因为调控 Wnt/ERK 多信号途径中的串话关键点。实验结果显示，火针可提高脊髓损伤大鼠神经功能评分，保护神经元、促进神经元修复；体内和离体实验显示火针可调控Wnt/ERK多信号通路关键靶点Wnt、β-cantenin、Gsk3β、ERK1/2、ngn1、cyclind1基因及蛋白表达，促进nestin和NSE阳性细胞表达；同时，慢病毒转染实验显示，Gsk3β基因沉默后，各组tubulin、β-cantenin、GFAP蛋白及基因表达较转染前均明显降低，但火针对上述基因及蛋白的调控作用仍优于模型组。 实验结果表明，火针可改善脊髓损伤大鼠神经功能缺损，通过调控Wnt/ERK多信号通路关键靶点基因及蛋白表达，促进内源性及离体神经干细胞增殖、向神经元及少突胶质细胞定向分化，而火针疗法改善脊髓损伤大鼠神经功能缺损的机制可能是通过调控Wnt/ERK串话关键点GSK3β，从而调控Wnt/ERK多信号通路，促进内源性神经干细胞增殖及向神经元定向分化。 本研究为火针疗法治疗中枢神经损伤性疾病提供了实验依据，可促进临床上火针疗法对治疗脊髓损伤等疾病的应用和推广。