椎间盘内源性修复失效机制及髓核原位再生研究

The failure of endogenous repair is the main cause of intervertebral disc (IVD) degeneration. Both foreign and our latest studies suggest that IVD stem cells play an essential role in IVD endogenous repair. However, it’s the key issue that how to maintain the sufficiency and vitality of stem cells under the adverse microenvironment comprising pressure and hypoxia..The study is based on our long-term researches on the mechanism and regeneration of IVD degeneration. Taking the IVD stem cells as the breakthrough point, with the help of the self-designed controllable axial loading induced IVD degeneration model in vivo and the self-designed controllable air-pressure cell culture apparatus in vitro, we will make further studies on the influence of adverse microenviroment on the biological behavior of IVD stem cells, such as migration, homing and death, as well as discuss the relationship and crosstalk of autophagy, apoptosis and necrosis of IVD stem cells, so as to clarify the molecular mechanism of fail of endogenous repair during IVD degeneration and to find the key regulatory target from the complicated network. Following that, we will pertinently design self-assembling peptide nanofiber scaffolds with multiple bio-signals in order to induce the migration, homing and differentiation of stem cells, contributing to the in situ regeneration of nucleus pulposus..The study will enrich the theories of degeneration and regeneration of IVD. It will provide a new strategy of biological therapy for IVD degeneration.

内源性修复失效是椎间盘退行性变的主要原因。国外及我们最新研究提示，椎间盘干细胞在椎间盘内源性修复中发挥重要作用。如何在椎间盘内压力低氧等不利微环境下维持数量足够、活性良好的干细胞是亟待解决的关键问题。.本项目拟在长期从事椎间盘退行性变机制和修复再生研究的基础上，以椎间盘干细胞为切入点，利用前期发明的椎间盘在体可控轴向加压模型和可控气压加压细胞培养装置，深入研究盘内压力低氧微环境对椎间盘干细胞迁移、归巢、死亡等生物学行为的影响；探讨椎间盘干细胞自噬、凋亡、坏死性凋亡的相互关系及串扰影响，阐明椎间盘退行性变过程中内源性修复失效的分子机制，从复杂网络中发现关键调控靶点；同时针对性设计一种携带多重生物信号的自组装多肽纳米纤维凝胶材料，多方位、协同、序贯诱导椎间盘干细胞向盘内迁移、归巢及定向分化，提高其质和量，从而促进髓核原位再生。.本项目将丰富椎间盘退行性变及修复的理论，为其生物学治疗提供全新策略

内源性修复失效是椎间盘退行性变的主要原因，椎间盘干细胞在椎间盘内源性修复中发挥重要作用。本课题组前期研究发现，压力等不利微环境是影响内源性干细胞生物学行为的重要因素，过度压力诱导髓核干细胞发生凋亡及坏死性凋亡，进而导致椎间盘衰老及退变过程中髓核干细胞的耗竭。在前期研究基础上，我们深入探究了椎间盘退变中髓核干细胞耗竭的具体分子机制和调控靶点。我们研究发现，在椎间盘退变过程中，低氧诱导因子1A(HIF1A)通过激活自噬减轻过度压力诱导的髓核干细胞的凋亡，而HIF1A-BNIP3-ATG7途径介导了低氧压力条件下髓核干细胞的自噬激活，从而增强了髓核干细胞对过度压力负荷的抵抗能力。我们的研究结果表明低氧微环境是保护内源性干细胞数量及活性的关键因素之一。抑制椎间盘内氧浓度的增高，调控髓核干细胞的HIF1A水平可能为椎间盘内源性修复创造有利条件。同时，我们发现抑制热休克蛋白90（HSP90）通过降低RIPK1、RIPK3、MLKL的稳定性及活性减轻压力诱导的髓核干细胞坏死性凋亡，并能够抑制线粒体功能紊乱及氧化应激减轻压力诱导的髓核干细胞凋亡。另外，我们发现环孢素A、雌二醇、葛根素等也可减轻压力诱导的髓核干细胞凋亡。更重要的是，我们针对性地设计并制备了髓核和纤维环脱细胞基质水凝胶以强化椎间盘内源性修复、促进髓核原位再生。我们研究发现脱细胞的髓核水凝胶和纤维环水凝胶可以分别诱导人骨髓间充质干细胞向髓核样细胞和纤维环样细胞分化，进一步研究证实Yes相关蛋白1(YAP1)参与了髓核及纤维环脱细胞基质水凝胶对人骨髓间充质干细胞组织特异性分化的调控。同时，体内实验也证实脱细胞的髓核水凝胶和纤维环水凝胶分别促进了髓核退变模型和纤维环缺损模型的体内再生。本课题的发现不仅从分子及细胞层面丰富了椎间盘退行性变及修复中髓核干细胞耗竭的理论，而且为椎间盘退变性疾病的生物学治疗提供全新策略。