TGF-β/AMPK信号通路介导马凡综合征的血管干细胞早衰及对其进行调控的治疗作用研究

The loss of vascular smooth muscle cells (VSMCs) is the major histopathological features of vascular media injury in MFS. Vascular wall stem cells (VSCs) have the potential to differentiate into VSMCs to maintain the self-renewal of vascular parietal cells and repair after injury. Our preliminary study showed that VSCs isolated from MFS exhibited the characteristics of senescence including decreased cell number, migration and proliferative capacity and increased cell size and mitochondrial fusion. Moreover, TGF-β which regulates cell senescence was greatly enhanced whereas p-AMPK which regulates mitochondrial fusion and fission was significantly reduced in VSCs from MFS. Based on the above observation, we hypothesized that TGF-β can induce mitochondrial fusion through regulating AMPK signaling pathway, leading to premature senescence of VSCs in MFS. Subsequently, the senescent VSCs are unable to complete tissue renewal and self-reinforcement of vascular wall, and eventually lead to various degenerative lesions of arterial wall. Here, we will use a series of molecular methods to explore the potential mechanisms of TGF-β/AMPK mediating mitochondrial damage induced the senescence of VSCs in MFS in vitro and in vivo. We will also test the beneficial effects of TGF-siRNA treated MFS-VSCs on aortic lesions in mice model of MFS. This study will provide theoretical and experimental basis of VSCs based therapy for prevention and treatment of the disease of large vessels and exhibit the important academic and clinical significance.

血管平滑肌细胞(VSMCs)丢失是马凡综合征(MFS)患者血管中膜损伤的主要病理特征。血管壁干细胞(VSCs)具有向VSMCs分化的潜能，维持血管壁细胞的自我更新与损伤后的修复。我们的研究表明，MFS的VSCs数量减少、体积变大、迁移和分化能力显著下降、线粒体融合变长，早衰特征明显；在VSCs中，与调控早衰相关的TGF-β明显升高，而与调节线粒体裂变融合相关的p-AMPK明显下降。可以假说：TGF-β通过调节AMPK信号通路诱导线粒体融合、引起MFS中VSCs的早衰、血管壁的自我更新不足，而导致动脉壁的退行性变。本研究体内外两个层面来探讨TGF-β/AMPK介导线粒体损伤和VSCs早衰的信号机制；并以TGF-β为干预靶点，通过siRNA转染技术处理MFS-VSCs，观察其对MFS动物模型主动脉病变的防治作用。研究结果可为VSCs在大血管疾病的防治提供实验基础，具有重要的学术价值和临床意义。

马凡综合征（MFS）血管病变是导致死亡的重要原因，其病理主要表现为中膜平滑肌细胞丢失，取而代之的为空泡样改变，胶原沉积。血管壁干细胞（VSCs）分布于血管外膜，在血管损伤后可分化为血管平滑肌细胞而起到损伤后修复作用。但为何MFS患者主动脉的VSCs无法发挥修复作用，导致疾病进展甚至死亡，其中机制还有待阐明。本研究分别选取器官捐献供体以及行主动脉手术的MFS患者主动脉组织，原代培养及免疫磁珠法筛选出c-kit+-VSCs，并鉴定比较其表型特征及细胞功能。发现与Control-VSCs相比，MFS-VSCs出现细胞衰老，表现为细胞体积增大，SA-β-gal阳性率高，衰老相关蛋白p53和p21表达增多。而且MFS-VSCs增殖、迁移能力降低，细胞衰老明显。同时我们对两组细胞进行转录组水平测序，结果显示几个与细胞过程相关的通路，包括细胞周期和细胞衰老，在MFS-VSCs中显著富集。而且TGF-β1在MFS-VSCs中的表达水平明显高于Control-VSCs。用TGF-β1处理Control-VSCs可诱导其衰老，并且线粒体表现为融合增多裂变减少。同时MFS-VSCs线粒体活性氧产生增多，当加入活性氧去除剂，或者抑制线粒体融合蛋白表达后，可缓解由TGF-β1诱导的细胞衰老。进一步对上游调控通路进行分析，发现AMPK信号通路介导了TGF-β1诱导的线粒体融合。TGF-β1可抑制AMPK信号通路，激活这一通路可使TGF-β1诱导的细胞衰老缓解。综上得出本研究的结论，MFS患者中较高浓度的TGF-β1通过抑制AMPK信号通路导致线粒体形态改变，使线粒体活性氧增多，氧化应激增强，激活衰老相关蛋白过表达，从而导致细胞衰老，功能下降。本研究探讨了TGF-β1介导的MFS-VSCs早衰机制，揭示了MFS患者血管稳态降低的原因，为临床早期干预MFS主动脉病变提供新的治疗靶点和理论依据。