内皮细胞外泌体通过TRAIL/PI3K/Akt通路调控内皮祖细胞迁移和血管化的作用机制研究

Angiogenesis is a key point in tissue reparation and reconstruction. The migration and angiogenesis of endothelial progenitor cell (EPC) play a vital role in this part. Various factors have been confirmed to be related with migration and angiogenesis of EPCS, such as VEGF、mRNA. In the previous study, we found: 1) in vascular injury site, endothelial cells (ECs) can secrete exosomes and co-culture of exosomes derived from endothelial cells and EPCs can enhance the ability of migration and angiogenesis of EPCs; 2) The expression of exosomal lncRNA NEAT1 derived from endothelial cells was enhanced; 3) TNF-related apoptosis inducing ligand (TRAIL) can induce the migration and angiogenesis of EPCs. Besides, PI3K/Akt signaling pathway has been reported to be a critical point in angiogenesis. On the basis of the previous study and literature, we hypothesize that exosome derived from endothelial cells regulating migration and angiogenesis of endothelial progenitor cells via TRAIL/PI3K/Akt signal pathway. The aim of the project is to investigate in depth the effect of exosome derived from endothelial cells on vascular repair and the mechanism involved to provide a novel therapeutic strategy.

血管再生是组织修复重建面临的关键问题之一，而内皮祖细胞（endothelial progenitor cells，EPCs）的迁移和血管化是血管再生的关键。已有多种因素被证实参与了EPCs迁移及血管化，如VEGF、mRNA等。前期研究中，本课题组发现1）氧化应激刺激后的内皮细胞周围出现外泌体样结构，外泌体与EPC共培养后能增强EPC的迁移和血管生成的能力；2）外泌体中lncRNA NEAT1表达明显增高；3）肿瘤坏死因子相关凋亡诱导配体(TRAIL)能增强EPCs的迁移和血管生成的能力。文献报道PI3K/Akt信号通路在血管形成中发挥重要作用。结合预实验，我们提出如下假设“内皮细胞外泌体NEAT1通过TRAIL及其下游的PI3K/Akt信号通路调控EPCs的迁移和血管化。”本项目拟通过深入探讨内皮细胞外泌体在血管再生中的作用及相关分子机制，为组织修复提供新方向。

皮瓣移植广泛用于组织器官的重建，确保皮瓣存活的前提是有足够的血供。本研究探讨暴露于氧化应激的人脐静脉内皮细胞分泌的外泌体对内皮祖细胞参与血管生成的潜在作用机制及其对皮瓣存活的影响。用不同浓度的H2O2处理HUVECs，建立氧化应激模型。超速离心获取经H2O2处理的HUVEC分泌的外泌体（H2O2-HUVEC-Exos）和未经H2O2处理HUVEC分泌的外泌体（HUVEC-Exos）。利用透射电镜、粒径分析、蛋白免疫印迹实验等检测外泌体的理化性质。体外利用H2O2-HUVEC-Exos和HUVEC-Exos进行EPC的CCK8、成管及迁移实验，探究这两种外泌体在体外的促血管生成能力。微阵列分析用于揭示H2O2-HUVEC-Exos和HUVEC-Exos中差异表达的长链非编码RNA (lncRNA)。此外，采用基因沉默和蛋白质印迹来确定EPC中Lnc NEAT1下游的作用机制。此外，通过大鼠皮瓣模型探究外泌体在体内对皮瓣存活的影响。用100 μmol/L H2O2刺激HUVECs 12 h以建立氧化应激模型。与HUVEC-Exos 和对照组相比，H2O2-HUVEC-Exos能明显促进EPCs的增殖、成管和侵袭能力。H2O2-HUVEC-Exos中的Lnc NEAT水平显着提高并激活Wnt/β-catenin信号通路。相比之下，Lnc NEAT1的敲低导致 EPC 的生物学行为发生显着变化，并降低了Wnt/β-catenin 信号通路相关的蛋白质表达水平。与PBS和HUVEC-Exos相比，动物体内实验显示H2O2-HUVEC-Exos可以增加皮瓣的存活面积、血管数目和归巢的EPC数目。因此，H2O2-HUVEC-Exos 的应用有望作为提高随机皮瓣存活率的替代疗法。