Wnt/β-catenin信号通路在noggin基因转染NSCs移植脑缺血再灌注损伤中的调控机理

Cerebrovascular disease frequently occuring in central nerve system causes serious damage to human life, but the mechanism underlying neurological repair and regulation after injury remains incompletely known. Current evidences showed neural stem cells (NSCs) not only emerged as potential seeding cells for the cerebral ischemia reperfusion injury (IRI), but could acte as carrier for gene therapy. Wnt/β-catenin signal was one of the important ways for proliferation and regulation of NSCs. Our previous study has confirmed noggin gene was implicated in promoting proliferation of hippocampus NSCs and improving behaviors, suggesting NSCs proliferation may be associated with Wnt/β-catenin signaling pathway. In this study, we attempt to deliver noggin gene into cultured rat NSCs. Transplantation of transected NSCs into the rat lateral ventricle after IRI with in situ expression of noggin gene promotes endogenous neurogenesis, promotes the differentiation of neurons and oligodendrocytes，and compensates the neuron loss after injury. We examine the proliferation, differentiation and migration of transfected NSCs in the ischemic penumbra, meanwhile detect Wnt-1、Axin and BMP expression before and after transplantation, to explore the mechanism that Notch signaling regulates transfected NSCs proliferation, differentiation and migration, as well as to pave the way for clinical application of NSCs transfected with noggin gene in IRI treatment.

脑血管疾病是严重危害人类生命与健康的常见病和多发病，但其损伤后神经功能的修复与调控机理尚不清楚。神经干细胞(NSCs)不仅可作为治疗脑缺血再灌注损伤(IRI)的种子细胞，还可作为基因治疗IRI的很好载体。Wnt/β-catenin信号是NSCs增殖调控的重要途径之一。前期我们已证实，noggin能促进衰老小鼠海马NSCs的增殖与增强记忆力。据此，我们推测NSCs增殖可能与Wnt/β-catenin信号通路的调控有关。本课题拟将noggin 基因转染体外培养的大鼠NSCs，再经侧脑室移植入大鼠脑内。通过noggin 基因的原位表达，促进内源性神经的发生、补充损伤丢失的神经元。观察基因修饰NSCs在海马与缺血半影区的增殖、分化与迁移规律；最后检测Wnt/β-catenin信号通路在对noggin修饰NSCs增殖、分化与迁移的调控机制，为临床应用NSCs移植治疗IRI调控提供理论依据

缺血性脑中风是严重威胁人类健康的常见病，治疗原则是及时恢复缺血区的血供，挽救受损的神经元。但是缺血区再次血流灌注后往往会造成神经元的进一步损伤即脑缺血再灌注损伤，其损伤机制复杂而未解决。近年来国内外研究表明，神经干细胞（NSCs）移植对于脑缺血后的神经功能改善有一定疗效。由于忽视了局部微环境，导致移植NSCs存活率低、增殖分化少等诸多问题尚未解决；难以获得长期稳定的疗效；另外NSCs移植后增殖、分化的调控也是当今研究的热点。故本项目共分为4部分：1. noggin基因重组腺病毒的构建及鉴定; 2.大鼠神经干细胞分离、培养及鉴定; 3. noggin基因转染大鼠NSCs的表达及鉴定; 4. noggin基因转染NSCs的移植与Wnt/β-catenin信号对NSCs的调控。研究结果：1.经酶切鉴定、基因测序和PCR结果证实含noggin基因重组腺病毒载体构建成功；2.成功分离、培养了大鼠海马NSCs，并进行了noggin重组腺病毒载体NSCs的成功转染；3.线栓法建立局灶性脑缺血再灌注模型，成功率达80%以上；4.立体定向NSCs侧脑室移植，发现NSCs移植可显著改善脑缺血后受损的神经功能，减少梗塞体积、炎症反应与神经元的凋亡；5.荧光双标、RNA干扰等方法检测，发现激活Wnt/β-catenin信号通路后可以促进β-连环蛋白，GSK-3，wnt3a等重要信号分子上调noggin基因表达；促进移植NSCs向受损脑区迁移、分化；反之，移植NSCs向受损脑区迁移、分化受到抑制，表明Wnt/β-catenin 信号通路参与了noggin基因转染NSCs对受损脑区的修复，证明noggin基因修饰的NSCs移植治缺血性脑卒中是可行的，但远期疗效与用于临床应用还需要进一步探讨。本项目这些重要结果和关键数据为noggin基因修饰NSCs移植治疗缺血性脑卒中奠定了坚实基础，也为调控NSCs移植治疗缺血性脑卒中提供了重要线索。目前发表SCI论文3篇、北大核心论文4篇；重要学术会议交流4次； 6名研究生中3名已取得硕士学位并获得校级优秀论文二等奖1项、三等奖2项。