缓激肽及其B2受体活化调节内皮祖细胞衰老的作用及机制研究

Stress-induced premature senescence (SIP) could impair endothelial progenitor cells (EPCs) function and proliferative capability, there is a negative correlation between EPCs function and cardiovascular risk factors. Bradykinin protects endothelial cells from SIP, however, the exact molecular mechanism of SIP and their role in EPCs dysfunction still needs to be elucidated. Our preliminary data showed BK inhibited H2O2-induced EPCs senescence through B2 receptor (B2R) activation, the level of RB1 transcripts was decreased by approximately one hundred and seventy-six fold by human cellular senescence PCR array. Here, we describe a novel mechanism of BK/B2R signal pathway activation leading to EPCs antisenescence. EPCs will be studied using Adenovirus transfection, RNA interference, real time PCR, Westernblotting, human cellular senescence PCR array, cell cycle control phospho antibody array and signal transduction inhibitors and agonists. In db/db diabetic mice, Ad.B2R modified EPCs will be loaded with near-infared dye and image in a wire-induced carotid injury model over a time course of 2 weeks using Maestro in vivo imaging system, to observe EPCs survival and senescence. We try to clarify the mechanism of BK/B2R activation EPCs inhibited SIP through B2R/ PI-3K-Akt /cyclinD/E or EGFR transactivation /cyclinD/E/RB pathway. Identification of the exact mechanisms of BK/B2 activation inhibition EPCs senescence may have important implications on the development of novel molecular therapies for the prevention of coronary heart disease and diabetic angiopathies

应激诱导细胞衰老（SIP）致内皮祖细胞（EPCs）数量及功能下降，与心血管病危险因素负相关。缓激肽（BK）B2受体（B2R）活化抑制内皮细胞SIP，其机制不明。我们预试验新发现BK/B2R活化抑制EPCs SIP，其机制与抑制视网膜母细胞瘤基因（RB）功能相关。申请者拟使用B2R激动剂、抑制剂、B2R腺病毒转染，siRNA干扰，RNA及蛋白芯片筛选等方法，研究BK/B2R活化抑制EPCs SIP。使用信号通路中多个调控单元的阻断剂和激动剂对相关的信号通路进行研究。使用糖尿病小鼠颈动脉内膜损伤模型，移植B2R高表达EPCs，活体成像，动态观察EPCs在损伤内膜的存活及衰老，阐明BK/B2R活化抗衰老通过B2R/PI-3K/Akt/周期蛋白/RB和/或B2R转激活EGFR/周期蛋白/RB途径。通过BK/B2R轴调节EPCs抗SIP，对改善EPCs功能, 治疗冠心病、糖尿病血管病变具有重要意义。

应激诱导细胞衰老（SIP）致内皮祖细胞（EPCs）数量及功能下降，与心血管疾病危险因素负相关。缓激肽（BK）及B2受体（B2R）参与了SIP过程对EPCs功能调节。本课题通过衰老芯片筛选SIP相关靶蛋白，运用分子生物学、细胞生物学及病理学等方法，对BK-B2R参与调控SIP诱导EPCs衰老的分子机制进行了深入研究。研究发现B2R的表达在糖尿病及妊娠性糖尿病患者外周血及脐血EPCs显著下降，与氧化应激（ROS）负相关。在H2O2诱导EPCs衰老中发现，BK通过B2R依赖的AKT活化抑制视网膜膜细胞瘤蛋白（RB）及EGFR活化抑制RB、抑制ROS导致的EPCs衰老。在高糖诱导EPCs衰老研究中发现BK可明显改善高糖所致EPCs损伤，增加EPCs增殖、克隆、NO分泌量等，高糖抑制EPCs端粒酶活性，BK可显著增加EPCs线粒体和细胞核端粒酶表达，提高端粒酶活性，BK通过B2R活化激活AKT-eNOS促进端粒酶逆转录酶功能抑制高糖诱导的EPCs衰老。课题组通过对组织激肽释放酶缓激肽系统（KKS）中组织激肽释放酶结合蛋白（KS）的开创性研究，发现KS在冠心病患者血浆显著下降，其下降程度与病变程度负相关。通过腺病毒转染KS能抑制小鼠颈动脉斑块的形成，抑制剂的阻断研究显示，KS通过SIRT1-eNOS途径抑制ROS的形成，通过KLF4途径促进M2细胞转分化抑制炎症，该研究的结果揭示了KKS在心血管保护作用的新功能。本课题组的研究为深入探讨KKS在心血管疾病中的保护作用，阐明BK/B2R 轴调节EPCs 抗 SIP的分子机制奠定了坚实的工作基础。对改善 EPCs功能, 治疗冠心病、糖尿病等血管病变提供了重要的依据。项目资助发表SCI收录论文10篇，待发表3篇，中文核心期刊1篇。培养博士生4名，硕士生2名，其中2名已经取得博士学位，2名博士在读，2名硕士在读。项目投入经费73万元，支出57.3511万元，各项支出基本与预算相符。剩余经费15.6489元，剩余经费计划用于本项目研究后续支出。