Th17/IL-17在血管内皮细胞衰老进程中的作用及机制研究

Senescence of Vascular endothelial cells (VEC) may play an important role in the development of cardio- and cerebro- vascular diseases, especially in atherosclerosis (AS). However, the mechanism of senescence occurrence and regulation in VEC is not clear. It has been reported that the levels of Th17 cells and related cytokine IL-17 were significantly higher in healthy elderly than in youth.Our preliminary studies suggested that IL-17 can promote VEC senescence. Expression of senescence-associated secretory phenotype(SASP) in aging VEC is increased. VEC senescence is closely related to NF-kB and MARK. Therefore, we proposed the following hypothesis: In the aging process, upregulated IL-17 receptor (IL-17R) binds with IL-17 secrected from Th17, resulting activation of NF-κB and MAPK, and further activation of p53/Rb pathway which induces VEC senescenes. SASP elevation may increase Th17/IL-17 expression and promote VEC senescence. Our studies strive to demonstrate the hypothesis from "overall-tissue-cell -molecular" level：1)the effect of Th17 and its secreted IL17 on VEC senescence; 2)the signaling pathways of Th17/IL17 on VEC senescence; 3)The effect of Th17/IL-17 on SASP in aging VEC and its molecular mechanism;4) discovery of novel markers related to scenescence in AS. In this study, health people and AS patients in different ages, aging mouse models and aging endothelial cells as well as intervention in intro will be used by the application of cell and molecular biology techniques, such as small interfering RNAs (siRNAs), flow sorting and so on. The outcomes of this study will provide new ideas and targets for prevention, diagnosis and treatment of diseases, such as AS.

血管内皮细胞（VEC）衰老在动脉粥样硬化（AS）等心脑血管疾病发生发展中发挥重要作用，但其发生和调控机制尚不完全清楚。文献报道Th17及IL-17在健康老年人体内水平均明显升高；申请人前期研究表明，IL-17可促进VEC衰老，且衰老的VEC表达衰老相关分泌表型（SASP）增多；预实验结果提示NF-κB和MAPK与VEC衰老密切相关。据此推测"在增龄过程中，Th17水平升高，其分泌的IL-17与IL-17R结合后，激活VEC NF-κB、MAPK；继而活化p53/Rb通路，引起VEC衰老；其SASP增多又可进一步上调Th17/IL-17表达，并促进VEC衰老。"本课题拟以不同年龄段健康人群及AS患者、自然衰老小鼠模型和衰老内皮细胞为研究对象，应用流式分选、siRNA等技术，从整体-组织-细胞-分子等层次探讨Th17/IL-17在VEC衰老中作用的分子机制，为AS等疾病的预防和诊疗提供新思路。

血管内皮细胞（VEC）衰老在动脉粥样硬化（AS）等心脑血管疾病发生中发挥重要作用，但其调控机制尚不完全清楚。文献报道和前期研究表明，IL-17 可促进 VEC 衰老，信号通路NF-κ B 和 MAPK 与 VEC 衰老密切相关。本研究以不同年龄段健康人及 AS 患者、自然衰老小鼠模型和衰老内皮细胞为研究对象，探讨Th17/IL-17 在 VEC衰老中的作用机制。研究表明，健康老年人Th17/IL-17表达水平显著高于健康青年和中年人，且与血管内皮衰老标志物和AS生化标志物水平显著相关，可能成为VEC衰老和AS相关疾病诊断和预后评价的重要指标。动物实验发现，老年鼠动脉IL-17、IL-17RA和内皮衰老指标VCAM-1表达水平也明显高于青年鼠和中年鼠。老年鼠原代VEC 衰老染色阳性率、P16和Rb等衰老蛋白表达水平明显增高。通过基因测序和蛋白芯片研究发现了青年鼠和老年鼠内皮细胞差异基因和血清差异蛋白。IL-17-/-鼠动脉VCAM-1表达、原代VEC 衰老染色阳性率、衰老蛋白表达水平和NF-κB、JNK、P38等通路蛋白表达和磷酸化水平也明显低于野生鼠。在体内干预实验中，Th17细胞和IL-17蛋白干预组鼠动脉IL-17RA和VCAM-1表达水平、原代VEC衰老染色阳性率、衰老蛋白表达和上述通路蛋白表达和磷酸化水平均明显高于对照鼠，而IL-17抗体干预组上述指标水平均明显低于对照鼠。细胞实验发现在一定范围内，随着IL-17浓度增加，内皮细胞IL-17RA表达水平、衰老染色阳性率均升高，衰老蛋白表达水平、培养上清SASP因子浓度明显增高。一定浓度IL-17作用24h后，NF-κB、P38和JNK的蛋白表达水平和磷酸化水平均明显高于未处理组；在分别加入以上通路抑制剂作用后，各组衰老蛋白水平均明显减低。IL-17与IL-17RA基因沉默的VEC作用48h，发现衰老染色阳性率、衰老蛋白表达等均无明显变化。另证实SASP因子IL-1β和IL-6可明显促进Th17细胞分化，加速VEC衰老进程。以上结果表明Th17/IL-17可通过与VEC表面IL-17RA结合，激活NF-κB、P38和JNK信号传导通路，活化p53/p19/p21和p16/Rb衰老途径，促进VEC衰老和功能紊乱。该研究为 AS 等疾病的诊疗和预后判断提供新思路。