电针促进脑缺血后内源性NSCs增殖分化的机制研究

Two acupoints, "QUCHI"(LI11) and "ZUSANLI"(ST36) were considered the most effective prescritions and commonly used for electroacupuncture in the treatment of ischemic stroke. Our preliminary data demenstrated that electroacupuncture with these two acupoints can significantly improve nerve stem cells (NSCs) proliferation and differentiation after cerebral ischemia. Also, the number of active proliferate NSCs, the quantity and ratio of NSCs differentiate into neurons or glial cells can directly influent the rehabilitation of the patients suffered ischemic stroke. It was known that NSCs proliferation and differenciation after cerebral ischemia were regulated by Wnt、Notch and Shh signal pathway. To further explore whether electroacupuncture can regulate the number of proliferative NSCs and the ratio of NSCs differentiation through the modulation of Wnt、Notch and Shh signal pathway，our project will apply "QUCHI" and "ZUSANLI" electroacupuncture to rat MCAO model and detect NSCs proliferation and the cell cycle with flow cytometry in order to identify the number and differential ratio of all types of nerve cell. Newly proliferated and static NSCs will be sorted to analyse the expression of related molecules within Wnt、Notch、Shh signal pathways in NSCs . The goal of this project is to elucidate the therapeutic mechanisms of "QUCHI" and "ZUSANLI" electroacupuncture in cerebral ischemia through treatment-induced NSCs proliferation and differentiation , and to provide the theoretical and experimental basis for clinical application of electroacupuncture.

在电针治疗缺血中风中，"曲池"、"足三里"穴是最有效、常用的组穴之一。课题组前期研究发现电针这两穴能有效促进脑缺血后神经干细胞（NSCs）增殖分化。现有研究表明NSCs的增殖数量和分化为神经元、胶质细胞的数量和比例直接影响缺血中风的康复，脑缺血后NSCs增殖分化受Wnt、Notch、Shh三条信号通路调控。为进一步探讨电针改善缺血中风与调控上述通路以及NSCs增殖分化的数量、比例之间的关系，本研究通过电针干预MCAO大鼠的"曲池"、"足三里"穴，用MoFlo XDP检测脑缺血后NSCs的增殖数量和细胞周期及其进一步分化为各类神经细胞的数量和比例，分选出新增殖NSCs和静止期NSCs，在细胞水平检测NSCs的Wnt、Notch、Shh信号通路相关因子的表达，阐明电针调控NSCs增殖分化治疗缺血中风的作用机制，为电针临床应用提供理论依据和实验。

本项目研究发现电针曲池、足三里可以有效降低神经功能缺损和减少脑梗死体积，改善缺血性脑卒中后运动功能。电针可促进内源性神经干细胞（NSCs）增殖和分化为各类神经细胞相关，Nestin/Brdu双阳性显示NSCs在缺血后被速度激活，第7天达到增殖高峰，提取原代NSCs发现其功能具有外排Hoechst 33342的能力；NSCs的分化表现为首先分化为星型胶质细胞，Brdu/GFAP双阳性随着电针干预时间而增加，在第14天趋于平台期，其次Brdu/DCX双阳性显示未成熟神经元在第14天开始出现，而Brdu/NeuN双阳性显示成熟神经元在第21天开始出现；进一步检测细胞增殖和分化相关蛋白CDK4、Cyclin D1、Ngn1和NeuroD的表达均升高。这些标志性蛋白的表达受到NSCs增殖分化Wnt、Notch、Shh三条信号通路的调控。电针治疗促进室管膜下区三条信号通路关键节点JAG1、NICD、Hes1、Hes5、Wnt1、β-catenin、Shh、Gli蛋白表达均升高。因此，电针可能通过Notch1、Wnt、Shh信号通路多靶点促进NSCs增殖分化，但信号通路关键节点分子在NSCs分化为各类细胞中的精确调控有待进一步明确。