神经肽物质P调控脊髓内源性神经干细胞增殖与分化机制的研究

Purpose: In this study, we will investigate whether substance P(SP) can enhance recovery from spinal cord injury (SCI) in mice through its unknown function of stimulation of endogenous neural stem cell proliferation,survival and differentiation after SCI. We will present the clue of development of new stem cell therapeutic target for spinal cord injury throughout determine the mechanism of SP stimulate the endogenous neural stem cells differentiation pathway...Contents: Spinal cord injury induces the progressive apoptosis of neuron and oligodendrocyte which results into demyelination and axon degeneration and eventually leads to spinal cord dysfunction. Recent studies noted that substance P (SP), a neuropeptide, plays an important role in the wound-healing process by recruiting bone marrow stem cells (BMSCs) to the injured tissue, the result reported at Nuture medicine(our lab, Nat Med. 2009，15，425-435). Our recent new findings suggest that SP can significantly improvement the functional recovery after SCI. That resulted by markedly alleviated neural cell apoptosis and extensively recruiting neuron like cells in the lesion cavity and then significantly increases neurotrophic factor (CNTF and NGF) with robust axonal outgrowth at lesion site. .Therefore, we will firstly using nestin-GFP transgenic mouse to examine the effect of SP on ependymal neural cells proliferation and differentiation after SCI and then using marker staining clarifies the neural cell phenotypes. .Next we will determine whether SP stimulation neural stem cells self-renewing and plasticity (neuron, astrocyte, oligodendrocyte respectively) from a freshly isolated nestin-GFP positive cells in spinal cord of mice..Finaly, we will determine the molecular mechanism of SP which has influence with endogenous neural stem cell proliferation and differentiation into mature neural cells by MAPK-Erk signaling pathways. Substance P is known to preferentially bind NK-1R and activate the G protein-coupled signaling pathway and the mitogen-activated protein kinase-extracellular signal related kinase (MAPK-Erk) pathway in spinal ganglion neurons, glioma cells and smooth muscle cells leading to cell proliferation...Expected Contribution: This proposal is a first study to investigate the role of SP in endogenous neural stem cell activation, which help to replenish cellular constituents lost by injury and allowd an effective development of neural stem cells based therapies of SCI.

脊髓损伤（Spinal cord injury, SCI）是一种严重的神经系统致残性疾病，损伤后神经细胞的凋亡及轴突的断裂成为临床治疗的一大难题。申请人在前期研究中发现，神经肽-P物质（Substance P, SP）可以减少神经细胞的凋亡、损伤节段空洞内细胞明显增加、增加神经营养因子(CNTF,NGF)的表达、促进了轴突的再生、使其下肢运动功能明显恢复。但是对于细胞数量增加的来源以及轴突的再生等机制仍不明确,可能参与调控内源性神经干细胞的增殖分化。拟在进一步的研究中采用nestin-GFP小鼠建立SCI，通过免疫组化、分子生物学检测等技术跟踪追查对体内外nestin-GFP细胞增殖、分化的影响，重点探讨SP是否直接作用于内源性神经干细胞的再生，旨在阐明SP调控内源性神经干细胞增殖分化的具体分子机制，为临床应用SP进行细胞治疗提供新的治疗方案，为神经退行性疾病新药开发奠定可靠的理论基础。

本研究利用Nestin-GFP转基因小鼠，探讨是否神经肽物质P（SP）参与动员内源性神经干细胞的增殖与分化，探索 SP 调控内源性神经干细胞增殖、分化的具体的分子机制是否通过调控JAK/STAT 和 MAPK 信号通路，同时关注促进和改善神经再生的方法，并且利用该转基因动物研究了Nestin阳性细胞在组织成体干细胞的维持，组织修复中的作用。.利用Nestin-GFP转基因小鼠动物脊髓损伤模型，发现静脉注射SP可以促进损伤修复，并在损伤区明显增加Nestin+阳性细胞。首先观察了内源性神经干细胞的增殖情况；通过Edu增殖相关染色发现，与PBS组相比静脉注射SP组脊髓损伤区Edu阳性细胞显著增多，尤其是Nestin+/Edu+细胞明显增加；细胞染色显示，在静脉注射SP组损伤区的头、中间、尾侧的Tuj1+/Edu+ 细胞均比PBS处理组增加3倍以上，且减少了GFAP+/Edu+细胞的数量。同时，体外实验结果显示，SP可以激活MAPK-Erk 信号通路，激活神经干细胞的增殖，促进神经元的分化，而对明显降低向GFAP的分化。提示，SP 可以通过激活MAPK-Erk信号通路动员内源性神经干细胞增殖，且促进其向神经元的分化，而抑制向星形胶质细胞的分化，从而加快损伤修复过程，为进一步利用SP动员内源性神经干细胞进行细胞治疗提供新靶点，为对中枢神经系统退行性疾病的新药开发提供可靠地理论依据。.将负载浓度梯度的NGF神经营养因子的生物支架移植于神经缺损损伤动物模型内，可以导向神经轴突生长方向，促进损伤两端轴突的生长速度，修复神经断端的神经再生及衔接。为方法为利用生物材料治疗神经损伤修复提供了重要的参考依据和新的治疗方案。.利用Nestin-GFP小鼠系统分析了Nestin除了神经系统外，在其它器官如睾丸，肾脏等组织表达特征，并且利用流式细胞技术分离获得的睾丸和肾脏组织Nestin-GFP阳性细胞具有肾间质干细胞和睾丸间质干细胞的特征，具有自我更新与多向分化能力，移植于体内促进病损组织修复。提示Nestin阳性细胞可能是包括神经系统在内的多个成体干细胞的共同标志，为进一步研究成体组织干细胞的发育起源、组织之间相关性以及稳定性维持等机制的研究提供了重要依据。