miR-25-3p 介导的老年血管生成障碍新机制研究

Incidence of lower limb ischemia increases in the aging population, which leads to high disability and mortality. Aging factor is usually ignored,which leads to unsatisfied current intervention, surgical and biological managements. Dysfunctional VEGFR-2 signaling has been well recognized to lead to impaired angiogenesis in aging population. Our pilot experiments showed decreased expression of miR-25-3p in aged endothelial cell (EC) lower expression miR-25-3p upregulates TULA-2 expression, which would lead to reduced phosphorylated Y323 of SYK, and consequently result in declined phosphorylated Y1175, and consequentlyimpaired angiogenesis mediated by VEGFR-2 signal pathway in aged vascular endothelial cells. We are going to employ dual-luciferase reporter gene assay, immunoprecipatition et al methods to illustrate the molecular mechanism of impaired angiogenesis which results from dysfunctional ageing vascular endothelial cell regulated by miR-25-3p and the potential mechanism of lower limb ischemia. Furthermore we are going to detect the protein expression in human ageing peripheral artery tissue, and use miRNA injection in vivo and limb ischemia salvage in mice to confirm the correlationship between miR-25-3p and impaired angiogenesis. This study will provide the novel insights to study the mechanism and the new therapeutic method of lower limb ischemia in aging population.

老年人下肢动脉缺血发病率、致残死亡率高；被忽略的年龄因素是目前介入、手术、生物学治疗效果不佳的重要原因。VEGFR-2信号传导通路功能低下是老年血管生成障碍的重要原因。前期人和动物研究发现老年血管内皮细胞（EC）表达降低的miR-25-3p，通过直接上调TULA-2、使SYK Y323去磷酸化，继而下调VEGFR-2 Y1175磷酸化，从而妨碍VEGFR-2信号通路介导的血管生成。拟采用双荧光素酶报告基因、蛋白免疫共沉淀等方法阐明miR-25-3p通过调控TULA-2／SYK／VEGFR-2信号通路，导致老年血管内皮细胞参与血管生成障碍的相关具体分子机制及其造成下肢缺血的内在机理；通过检测人类老年下肢缺血动脉组织标本，老年小鼠体内miR-25-3p基因注射及下肢缺血挽救实验，明确miR-25-3p和老年下肢动脉缺血疾病的临床相关性。本项目将揭示老年下肢动脉缺血发病新机制，为治疗提供新途径。

老年人的下肢动脉缺血性疾病的发病率和致残率较高。高龄导致的血管内皮细胞衰老、VEGFR-2信号传导通路功能低下是老年血管生成障碍的重要原因。本研究发现衰老的血管内皮细胞中表达降低的miR-25-3p，通过直接上调TULA-2的表达，使SYK Y323去磷酸化增加，继而降低下游VEGFR-2 Y1175磷酸化，从而妨碍VEGFR-2信号通路介导的血管生成。研究顺利完成了以下的主要内容：1.阐明了在人脐静脉血管内皮细胞( HUVECs )中，miR-25-3p 的直接靶基因是TULA-2，通过调节SYK/VEGFR-2信号通路，参与调控血管生成的分子生物学机制。2.进一步检测miR-25-3p及其下游蛋白信号在人体和C57 BL-6小鼠动脉组织标本中随年龄的表达变化，验证了miR-25-3p通过TULA2-SYK-VEGFR-2受体信号传导通路，在老年血管生成障碍中发挥重要作用。3.通过建立老年小鼠下肢缺血模型开展动物实验，在体内验证了提高miR-25-3p和TULA-2水平能够改善老年小鼠下肢缺血情况，明确了miR-25-3p在老年小鼠下肢缺血后血管生成中的作用。研究揭示了老年血管内皮细胞中miR-25-3p的低表达和TULA-2的上调与血管内皮功能老化的相关性及分子生物学机制，并在老年雄性小鼠体内过表达miR-25-3p，改善了老年小鼠下肢缺血后的血流和功能恢复，为治疗老龄下肢动脉缺血提供新的治疗途径。