白藜芦醇通过酪氨酰tRNA合成酶通路调控内皮祖细胞增殖分化的机制研究

Endothelial Progenitor Cells (EPC) are a population of stem cells that can reproduce, migrate and acquire mature endothelial phenotype. It is important to understand that medications and hormones have an effect on these cells transplantation in vascualr restenosis. This knowledge may help us to stimulate and maintain EPCs as well as planning future pharmacological interventions. The recent studies have demonstrated that resveratrol(RSV) increased endothelial progenitor cells numbers and migration and led to the up-regulation of some important genes like CD34, CD31, KDR and eNOS. It has been proved that RSV could prevent TNF-α induced endothelial cells injury and ROS production by SIRT1-NF-KB in our previous research. However, the mechanism is still under investigated. Recently, one study present a co-crystal structure of RSV bound to the active site of Tyrosyl tRNA Synthetase(TyrRS). RSV nullified the catalytic activity and redirected TyrRS to a nuclear function, stimulating NAD + dependent auto-poly-ADP-ribosylation of PARP-1, which may related to SIRT1-NF-KB pathway and led to the stimulation of eNOS/NO pathway. Co-IP and Laser Scanning Co-focal will be used to observe the interaction between resveratrol and TyrRS. Gene engineering will be used to explore the effect of TyrRS on EPCs proliferation and differentiation. This will provide a novel insight into the therapy of resveratrol on in-stent restenosis (ISR).

寻找促进内皮祖细胞（EPCs）的增殖与定向分化的药物成为EPCs移植治疗血管再狭窄的重要方法之一。新近研究发现白藜芦醇可促进大鼠体内 EPCs 的增殖迁移和内皮系基因的表达，改善内皮损伤修复，抑制再狭窄进展；我们前期实验证实白藜芦醇通过SIRT1 明显抑制 NF-KB 活性，保护内皮细胞功能。白藜芦醇具有酪氨酸样的多酚环结构，可与酪氨酰tRNA合成酶(TyrRS)结合，影响下游基因表达。故推测在EPCs上，白藜芦醇与TyrRS结合后进入核内，激活下游核糖聚合酶 PARP-1通路，或与SIRT1-NF-KB 信号通路产生联系，激活 eNOS/NO 途径，促进EPCs增殖迁移而再内皮化。本课题将结合免疫共沉淀，激光共聚焦等技术观察EPCs内白藜芦醇对 TyrRS 的影响，应用基因敲除等技术研究 TyrRS对EPCs的增殖与分化的作用机制，为白藜芦醇干预EPCs移植血管损伤后再狭窄的治疗奠定实验基础。

白藜芦醇具有促进 EPCs 体外增殖、动员和内皮系基因表达；白藜芦醇与酪氨酸的结构极为相似。本课题研究白藜芦醇与 TyrRS 之间的关系，探明白藜芦醇对 EPCs 增殖与定向分化内皮系的调节机制。本课题成功培养了大鼠骨髓中内皮祖细胞（EPCs），采用内皮表面标记物CD31、vWF、FLK-1等细胞表型和免疫荧光法鉴定，证实为内皮祖细胞，进一步验证白藜芦醇可增强内皮祖细胞增殖活性。本课题组在SD大鼠心肌梗死动物模型上，将EPCs注射至心梗心肌周围，预实验结果发现，内皮祖细胞归巢不佳，而且对心梗后血流动力学、心功能和重塑等改善不明显。调整研究计划后，发现白藜芦醇可抑制内皮细胞的炎症因子（活性氧、IL-6 和 MCP-1），GO功能分析提示白藜芦醇可能通过多条信号机制影响炎症因子和保护心血管系统；白藜芦醇抑制氧化应激与 SIRT1, 2, 3, 4, 5通路有关，但减轻 CD40 表达仅仅通过 SIRT1, 5途径。观察临床高血压患者，结果提示，收缩压水平和血清中性粒细胞明胶酶相关载脂蛋白（NGAL）间存在显著相关性， NGAL升高易导致早期肾损害。探明白藜芦醇可能通过SIRT家族多条途径，进而影响对内皮细胞的炎症机制，为白藜芦醇在临床上应用奠定实验基础。