缺血性脑卒中后内源性干细胞参与神经血管重塑的改变和表观遗传学调控研究

Ischemic stroke is one of the leading cause of mortality and morbidity in the world. Many survivors of stroke suffer from persistent sensorimotor problems. Our long-term goal is to develop new therapeutics that facilitate sensorimotor functional recovery from ischemic stroke by harnessing the potential of adult brain neural stem cells (NSCs) and peripheral blood bone marrow stem cells (BMSCs). Historically, it had been thought that the adult brain is entirely incapable of replacing neurons lost to injury or disease. However, in recent years, the adult human brain has been discovered to harbor NSCs that can produce both neuron and glia. Experimental data from animal stroke models indicate that these NSCs can respond in the period shortly after injury, increasing cell proliferation and producing new neurons. However, our understanding of how ischemia and peripheral stimulation rehabilitation therapy affect the adult stem cell populations – particularly at the chronic stages of stroke – is largely incomplete. In pursuit of our long-term goal, the objective of this project is to determine how these stem cells respond to stroke from early to chronic stages of recovery. Our central hypothesis is that ischemic stroke induces persistent changes to the NSC and BMSC populations, affecting neurovascular repair for a prolonged period of time. In this proposal, we demonstrate a genetic fate-tracing methodology suitable for following the fate of adult stem cells for long periods of time. Finally, we highlight our ability to acutely isolate NSCs and BMSCs from the mice for modern transcriptome analysis for the purpose of gaining new mechanistic insights into the chronic effect of stroke upon adult stem cells. With these integrated approaches, we propose experimental plans that will determine the chronic effects of ischemic stroke upon the stem cell populations of the adult mice. Such understanding is critical to guide the development of future therapies that involve endogenous stem cells and adult neurovascular repair.

缺血性脑卒中是世界范围内致死致残的重大疾病之一。幸存者大多存在永久感觉运动障碍。本研究致力于研发动员内源干细胞促进感觉运动功能恢复的新疗法。一直以来成体脑内神经元被认为不能新生。直到近年在人成体脑内发现了可分化成神经元和胶质细胞的神经干细胞。动物模型揭示这些神经干细胞在卒中后迅速增生、参与神经新生。而成体干细胞群如何在缺血性脑卒中恢复期及外周刺激康复治疗发挥作用的研究尚少。本研究旨在揭示成体干细胞如何响应卒中发生发展并参与神经功能恢复，着重探讨卒中可能引起的神经干细胞群和骨髓干细胞群的持久改变及对神经血管重塑等的影响。为此，将采用荧光示踪技术特异标记成体干细胞，并进行在体长期追踪研究。将对急性分离的成体干细胞进行转录组学分析，揭开缺血性脑卒中恢复期成体干细胞的继发性改变和分子机制。这将极大促进调动内源干细胞和成体神经血管重塑的治疗研究。

Wnt3a通过Wnt/β-catenin通路发挥重要神经保护作用。为了验证Wnt3a的神经保护作用，采取慢病毒感染原代培养的神经元过表达分泌型Wnt3a基因，经MTT试验验证在氧糖剥夺情况下Wnt3a可有效提高细胞活性。采取Wnt抑制剂XAV939，有效阻断Wnt/β-catenin，降低细胞活性，减弱神经保护作用。经离体细胞模型，选取有效浓度在局灶性缺血性脑卒中动物模型验证，白色区域显示脑梗死区域，Wnt3a经鼻内输送有效降低大脑梗死体积，XAV939有效阻止神经保护作用。采用Wnt3a经鼻内输送方法，同时证明有效降低脑外伤后脑组织受损占位体积。TUNEL荧光染色结合免疫荧光共标分析，绿色表示细胞死亡，结合特异性神经元标记物提示神经元死亡，Wnt3a经鼻给药组显著降低死亡神经元数。进一步研究揭示，Wnt3a处理激活的通路主要基于经典Wnt通路，即Wnt/β-catenin信号上调BDNF、GDNF、Neuroligin-3和VEGF等介导。这些分子具有重要的神经保护和促神经再生能力。在脑外伤模型下，Wnt3a可有效降低MMP-9和Caspase-3，提高Bcl-2，发挥抗凋亡作用，保护损伤后血脑屏障完整性。通过免疫荧光染色，观察到DCX阳性的神经前体细胞从SVZ向损伤周区迁移。Wnt3a处理组显著提高此群细胞的迁移距离。进而检测了损伤周区的神经再生。免疫荧光揭示Wnt3a显著提高新生神经元数量，联合Wnt/β-catenin抑制剂XAV-939和Dkk-1抑制了神经元新生。同时检测到Wnt3a显著提高新生血管内皮细胞的数量。采用流式细胞术分析，Wnt3a可联合SCF治疗，并发挥进一步提高外周血骨髓干细胞数量，以进一步促进神经再生、血管再生等过程。多普勒血流检测显示了Wnt3a明显改善卒中后血流恢复和感觉运动功能康复。Wnt3a促进神经前体细胞的治疗作用。经鼻内移植的iPS诱导分化的神经前体细胞联合经鼻内输送的Wnt3a重组蛋白，通过进一步提高BDNF和Ang-1，可进一步提高损伤周区内源性新生神经元数量。本项目建立了无创的颅内给药方法，并且证实Wnt3a重组蛋白通过Wnt/β-catenin信号调控损伤后神经前体细胞的增殖、迁移和神经血管重塑，深入了解β-catenin信号下游在中枢神经损伤修复中的机制和内源性干细胞在长期功能康复中的机制提供了理论基础。