表皮生长因子受体调控MPTP小鼠室管膜下区和纹状体内源性神经再生的机制研究

Parkinson’s disease (PD) is the second most common neurodegenerative disease, resulting in irreversible disrupted motor control which may become a source of disability. Nevertheless, there is currently no effective therapy that can treat PD with certainty apart from long-term administration of L-dopa concurrent with diminished effectiveness and deep brain stimulation with enormous cost. More recently, scientific interest in PD has mostly focused on endogenous neurogenesis. Our preliminary data showed that i) massive and prolonged reactive astrocytes (RAs) in the striatum induced by MPTP acquired the properties of neural stem cells (NSCs), and provided neuroprotection via regulating the host niche; ii) activation of RAs which could improve the niche depended largely on endogenous neurogenesis in the subventricular zone (SVZ). Notably, the mitogen-activated protein kinase (MAPK) signaling pathway mediated by epidermal growth factor receptor (EGFR) participates in the proliferation, differentiation, and migration of endogenous NSCs. However, further studies are needed to elucidate whether the RAs in the striatum originate from NSCs within the SVZ and the recruitment of RAs could be enhanced by activation of EGFR, which may significantly promote anatomical as well as functional restoration of dopaminergic system. Also, the molecular mechanisms underlying the process are not well understood. In the present study, we will carry out single cell isolation as well as single cell RNA-sequencing to extend our previous investigations on the relationship between active neural stem cells (aNSCs) within the SVZ and RAs in the striatum of MPTP mice, particularly the alterations in anatomy, behavior, and functional neuroimaging after intraventricular infusion of EGF. Additionally, the mechanisms of ERK/MAPK signaling pathway in the process of regulating RAs recruitment which could promote endogenous neuroplasticity will be further discussed. Hopefully, we expect to provide ample evidence for therapeutic strategy for Parkinson’s disease.

帕金森病（PD）的内源性神经再生备受关注。我们前期研究发现：MPTP小鼠纹状体大量激活态星形胶质细胞（RAs）形成并表现出神经干细胞（NSCs）特性，促进多巴胺系统再生修复；室管膜下区（SVZ）的ERK/MAPK信号通路与内源性神经再生关系密切，SVZ内源性神经再生能力的恢复促进纹状体RAs的形成。表皮生长因子受体（EGFR）介导的ERK/MAPK信号通路参与细胞增殖、分化、迁移等过程。但激活EGFR能否促进SVZ的NSCs迁移至纹状体形成RAs以改善MPTP小鼠的神经功能仍有待研究。本课题拟在前期实验基础上，利用单细胞转录组测序技术分析MPTP小鼠激活态神经干细胞（aNSCs）与RAs的相互关系；评估EGFR激活后，动物行为学、功能影像、组织病理方面的变化；本研究还将深入探讨EGFR介导ERK/MAPK信号通路调控内源性神经再生的分子机制。这将为优化PD的治疗策略提供更丰富的理论依据。

本项目在研究目标的指导下执行研究计划，开展表皮生长因子（epidermal growth factor, EGF）的小鼠脑室内注射，从行为学、影像学、分子蛋白、信号通路等方面分析实验结果，阐述内源性神经修复再生的潜在机制。此外，本研究还在干细胞体外培养分化、室管膜下区（subventricular zone, SVZ）与脑肿瘤发生方面取得了进展。本研究发现EGF脑室注射后动物运动能力、脑组织代谢明显改善，受损多巴胺系统显著恢复，证实EGF的神经保护作用，依赖于SVZ的表皮生长因子受体（epidermal growth factor receptor, EGFR）功能完整。EGF注射后SVZ可见大量激活态星形胶质细胞（reactive astrocytes, RAs）表现神经前体细胞（neural progenitor cells, NPCs）性质。转录组测序分析证实，EGFR下游的KRAS基因是调控的重要靶点之一，通过RAS/MAPK信号传导通路诱导产物磷酸化和级联反应，为疾病治疗确定潜在靶向药物并提高EGF敏感性提供理论依据。体外实验应用3D藻酸盐水凝胶培养系统进行小鼠SVZ的神经干细胞（neural stem cells, NSCs）的重编程，并诱导多潜能干细胞（induced-pluripotent stem cells, iPSCs）扩增分化为具备血管生成能力的内皮细胞（endothelial cells, ECs），建立体外稳定干细胞培养体系以期开展细胞治疗研究。本课题在前期实验基础上针对侵犯侧脑室SVZ的胶质瘤发生发展进行探讨，发现SVZ星形胶质细胞样细胞EGFR、CCN1基因显著差异表达，揭示共同差异基因富集于EGFR/KRAS/MAPK信号通路并在肿瘤中激活。转录组测序结果与既往文献及数据库比较获得NAMPT、FCGBP、C1QL2、DHRS9基因可能作为潜在胶质瘤生物标志物，均与肿瘤诊断和治疗及患者预后密切相关。基因表达情况有待进一步在SVZ样本验证，以期从内源性神经细胞异常增殖并诱导血管生成角度阐述胶质瘤的发生机制。

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