过表达S1PR3增强EPCs功能及促进损伤血管修复的作用研究

Dysfunction of the vascular endothelium is a vital factor in the pathogenesis of vascular disease. Endothelial progenitor cells (EPCs) differentiate into mature endothelial cells after vascular injury in an effective way of re-endothelialization. However, the mechanism of differentiation of EPCs remains uncertain that might effect the repair of endothelialization. Sphingosine-1-phosphate (S1P) is a bioactive lipid, which influences proliferation, differentiation, migration, and survival of endothelial cells, but also of smooth muscle cells or bone marrow cells through activation of the G protein-coupled S1P receptors. In the subject, we change the expression of S1PR3 on the EPC through the gene transfection, small RNA interference technology, measure the intracellular calcium concentration and the store-operated Ca2+ entry (SOCE), test the expression of Stim1 ORAI1 and TRPC 1,try to explain the mechanism of S1P how to impact the EPC's functons such as proliferation, migration, and angiogenesis. We then use the three-dimensional cell culture in vitro to clarify the interaction of EPC and vSMC. At last we use the carotid artery injury model to interpret the mechanism of S1P how to impact the EPC's functions including proliferation, homing and angiogenesis in vivo. After all we try to find a new way about how to repair the damage of blood vessels for the vascular disease and find some new strategies and drug targets about prevention and treatment for a artherosclerosis and PCI.

血管内膜损伤是多种血管性疾病发展的关键因素，内皮祖细胞（EPC）是损伤血管再内皮化的一个重要细胞来源，但是病理情况下，EPC生物学功能严重受损，不能保证内皮的修复。1-磷酸鞘氨醇（S1P）是鞘磷脂代谢过程中产生的一种重要的信号分子，S1P通过其受体S1PR3参与调节血管内膜损伤后修复的多个方面。我们拟采用S1PR3基因转染EPC，观测S1P对转染后EPC生物学功能的改善，阐明S1P调节EPC功能的机制；利用细胞共培养体系，观测EPC对平滑肌细胞的影响；最后建立小鼠颈动脉损伤模型，采用转染S1PR3基因的自体脾源性EPC移植，观察损伤动脉产生的S1P介导S1PR3过表达EPC靶向归巢，增殖，抗凋亡的作用；并观察归巢的EPC过表达S1PR3竞争性抑制局部S1P促vSMC增殖的作用。通过上述实验获得从多个靶点阻抑损伤动脉不良重构的方法。为防治AS、PCI术后靶血管不良重构提供新的治疗思路。

内皮祖细胞在血管内皮细胞的再生中发挥着重要的作用，参与血管的形成。1-磷酸鞘氨醇可以直接调节细胞的生物学功能，它和许多病理生理过程有关。1-磷酸鞘氨醇通过其受体3可以促进内皮祖细胞的功能。在这项研究中，我们证实，S1P可以诱导内皮祖细胞的增殖、迁移、血管生成，并且抑制H2O2诱导的内皮祖细胞凋亡。我们的研究结果表明过表达S1PR3的内皮祖细胞在血管损伤后14天，加速了血管再内皮化和抑制新生内膜的形成。此外，我们发现，过表达S1PR3的内皮祖细胞在早期小鼠颈动脉损伤后竞争性消耗S1P，促进了平滑肌细胞凋亡及抑制血管平滑肌细胞迁移的可能性很大。我们提供的体内和体外实验的证据首次表明，移植基因修饰的内皮祖细胞可以增强内皮修复和抑制新生内膜形成的能力，从而促进血管损伤后的修复并获得较好的抑制不良重塑的效果。