Rictor促进缺血后新生血管成熟作用机制研究

Novel strategies for promoting the formation and maturation of nascent vasculatures are becoming hotspots in studying ischemic diseases. Vascular endothelial cells play pivotal roles in the vasculogenesis and angiogenesis. Although the underlying mechanisms are still under debate, mammalian target of rapamycin (mTOR)-Rictor is currently considered as a key factor in regulating the functions of vascular endothelial cells. In our preliminary experiments, the hind-limb ischemia was performed in mice with a conditional knockout of Rictor gene. Our findings showed that compared with the control group the amount of the nascent vasculatures in the ischemic area were significantly increased in Rictor conditional knockout mice. However, an extremely inordinate arrangement of the endothelial cells in neoformative capillaries was also observed. Meanwhile, levels of some cytokines indicating the stablization and maturation of nascent vasculatures, such as platelet derived growth factor B, were markedly decreased. Therefore, we hypothesize that Rictor might play crucial roles in regulating the process of angiogenesis in ischemic diseases by promoting the maturation of nascent vasculatures. Base on the data from our preliminary experiments and other documented studies, the present project is designed to further investigate the regulatory effects of Rictor on the maturation of nascent vasculatures by performing the hind-limb ischemia in Rictor conditional knockout mice. In addition, in vitro studies will also be performed to evaluate the effects of Rictor on the functions and the tube formation of the vascular endothelial cells under the condition of hypoxia and hypoglycemia. Moreover, the differentiation and maturation of the endothelial progenitor cells will also be assessed after the application of the small interfering RNA (siRNA) to knock down the expression of Rictor. Finally, the mechanisms involved in the effects of Rictor on the maturation of nascent vasculatures are also to be investigated with the expectation to find the new therapeutical strategies to ischemic diseases.

目前促进缺血后血管新生已成为治疗缺血性疾病的研究热点。血管内皮细胞(endothelial cells，ECs)在血管新生过程中起着关键性的作用。mTOR-Rictor是调节ECs功能的重要因子,但其在血管新生过程中的作用机制尚不明确。我们通过构建Rictor条件性基因敲除小鼠，并进行下肢缺血实验后发现：Rictor敲除小鼠术后缺血区新生血管量虽有明显增加，但新生血管ECs排列极其紊乱，且血清中促进血管稳定、成熟的细胞因子量显著性下降。因此我们推测：Rictor可能通过促进新生血管成熟过程而在缺血性疾病的恢复中发挥重要作用。本项目基于现有研究成果，拟进一步利用Rictor敲除小鼠下肢缺血模型及在缺糖、缺氧下的ECs体外成管模型，从整体动物水平及细胞水平观察Rictor在缺血后新生血管成熟中的作用，以期为探寻促进缺血后成熟血管形成的新的治疗靶点提供理论依据。

血管内皮细胞在血管新生过程中起着关键性的作用。mTOR-Rictor是调节血管内皮细胞功能的重要因子，但仍需进一步研究其在血管新生过程中的作用及其机制。本研究从整体动物水平及细胞水平系统观察了Rictor在缺血及肿瘤发生后新生血管成熟中的作用。在正常状态及缺糖缺氧状态下，通过阻断Rictor或干扰其表达后，血管内皮细胞增殖、迁移、侵袭能力均显著性降低。同时血管内皮细胞所产生的反映血管成熟稳定的细胞因子及相关信号通路蛋白的释放和表达或活性显著性下降。通过在体干扰小鼠Rictor表达后，进行小鼠下肢缺血造模手术。激光散斑血流检测显示下肢血流恢复情况显著性低于对照组。同时免疫荧光检测发现术侧腓肠肌虽有较多新生血管出现，但新生血管内皮细胞形态不规则、排列紊乱。因新生血管的成熟与稳定还可能在恶性肿瘤的发生发展中起着重要的作用，本项目利用民族药进一步验证了通过促进新生血管的成熟与稳定对于缺血性疾病及肿瘤均可发挥显著性的改善作用。综上所述，本项目研究进一步验证了新生血管成熟过程在缺血性疾病及肿瘤的发生发展中起着重要的作用，同时发现了Rictor可能通过调节新生血管内皮细胞成熟及稳定，从而成为治疗缺血性疾病甚至肿瘤的新靶点。