Wnt/β-catenin通路调控脑缺血后NSCs增殖分化的机制及安宫牛黄贴穴位敷贴的干预作用

Ischemic Stroke is one of the world-wide hazards and unsolved disease.The proliferation and differentiation of NSCs mediated by Wnt/β-catenin pathway was involved in the important processes of happening and development of ischemia.Point Application treating for ischemia is the special therapy of Jin’s Three-Needle Therapy——the new Cantonese style of acupunture. In this study, Wnt/β-catenin signaling pathway regulating the proliferation and differentiation of NSCs was taking as the starting point,our purpose is to study the molecular mechanism of point application with Angong Niuhuang sticker repairing the ischemic brain damage,we will use the techniques of immunofluorescence, Western-blot, RT-PCR and MTT to make researches including of in vivo and in vitro experiments,choosing different differentiated NSCs cells (neural stem cell line C17.2) and focal cerebral ischemia model in rats as research object.It will first select the key factors related to NSCs' proliferation and differentiation of Wnt/ β -catenin signaling pathway,on this basis, it will further explore the mechanism of point application with Angong Niuhuang sticker repairing ischemic brain injury.This research is not only very important to deepen and improve the fundamental research of the mechamism on nerve regeneration of cerebral ischemia, but also helpful to clarify the targets and pathways of point application repairing ischemic brain damage.It will provide enough theoretical basis for using this method in clinical.

缺血性卒中是世界范围危害极大、亟待攻克的疾病，Wnt/β-catenin通路介导的NSCs增殖分化参与脑缺血发生、发展的重要过程，穴位敷贴防治脑缺血为岭南针灸新学派“靳三针”特色疗法。本研究以Wnt信号途径调控NSCs增殖分化为切入点，以安宫牛黄贴穴位敷贴修复缺血性脑损伤的分子机制研究为目标，拟从体外和体内两方面，以不同分化状态的NSCs（神经干细胞系C17.2）和大鼠局灶性脑缺血模型为研究对象，采用免疫荧光、Western-blot、RT-PCR、ELISA等技术，首先筛选出Wnt信号途径中与NSCs增殖分化相关的关键因子，在此基础上，再深入探讨安宫牛黄贴穴位敷贴修复缺血性脑损伤的作用机制。本研究对深化和完善脑缺血后神经再生的基础理论研究具有十分重要的意义，同时有助于进一步阐明穴位敷贴修复缺血性脑损伤的关键作用靶点和途径，为该法应用于临床提供充足的理论依据。

缺血性中风作为卒中的主要类型，其高发病率、高死亡率、高致残率使其成为世界范围危害极大、亟待攻克的疾病。近年来，在缺血性脑损伤研究领域，神经干细胞（Neural Stem Cells, NSCs）疗法备受关注。Wnt/β-catenin 通路被证实是NSCs增殖分化的关键调控环节。中医药在脑缺血防治中已凸显优势，穴位敷贴治疗脑缺血为岭南针灸新学派“靳三针”特色疗法，目前临床研究证明该法可显著改善患者的神经功能缺损程度。但由于缺乏系统研究，其具体的作用机制目前尚不清楚。. 本研究以Wnt/β-catenin 信号途径调控NSCs 增殖分化为切入点，以安宫牛黄贴穴位敷贴修复缺血性脑损伤的分子机制研究为目标，初步验证了Wnt/β-catenin重要分子Wnt-3、Dishevelled、β-catenin，及其下游靶基因GSK-3β、Survivin、Caspase-3、Bcl-2、Bax、P53 、VEGF均与NSCs 增殖分化密切相关；在此基础上，研究了Wnt/β-catenin通路参与穴位敷贴防治缺血性脑损伤的可能机制为上调核心分子Wnt3、Dishevelled、β-catenin，激活靶基因Survivin，促进Bcl-2表达，增加分泌型VEGF 蛋白含量，促进NSCs的增殖；下调抑制因子GSK-3β、Caspase-3表达，抑制Bax、P53表达，增加Bcl-2/Bax比值，提高大鼠学习记忆能力及其神经行为学评分，降低脑损伤。从而为穴位敷贴防治脑缺血的临床应用提供实验依据。. 如本疗法能在临床推广应用，不但具有社会意义，同时也必将带来巨大的经济效益，其形成的产业化前景非常乐观。