激活的滤泡辅助性T细胞调节内皮祖细胞TR3/Nur77-lncRNA-Meg3途径促血管新生的机制研究

Mechanism of orphan nuclear receptor TR3/Nur77’s participation in non-VEGF dependent angiogenesis is still elusive, and while new discovered long noncoding RNA-Meg3 was shown to play a key role in the activation of endothelial progenitor cells (EPCs) to lauch the angiogenesis process. Data from our previous clinical studies showed that numbers of follicular helper T cells and concentration of IL-21 in peripheral blood among patients with chronic total occlusion (CTO) were varied, which was signifcantly higher among patients with good coronary collateral circulation than those with insufficient collateral circulation. Expression of Nur77 on ischemic limb was significantly higher than that of control limb in diabetic mice models, in which lncRNA-Meg3 expression was uncovered an opposite trend. Based on the above all, a hypothesis was proposed that activated follicular helper T cells would promote the angiogenesis process of endothelial progenitor cells via activation of TR3/Nur77-lncRNA-Meg3 axis. In order to prove it, firstly, a case-control study with increased sample size will be conducted to investigate the exact relationships among circulating Tfh levels, activities of circulating EPCs and grades of collateral circulation; secondly, the involved molecular mechanisms will be explored in vitro experiment; and at last the mechanisms of activated Tfh promoting angiogenesis by regulating TR3/Nur77-lncRNA-Meg3 axis on EPCs will be investigated in cultured EPCs and transplantation of modified EPCs into diabetic mice model with hind limbs ischemia. Data of our study will not only benefit for uncovering the exact mechanism but also provide potential strategies for clinic.

孤儿核受体TR3/Nur77可介导非血管内皮生长因子依赖的血管新生，但机制不清，新发现LncRNA-Meg3是内皮祖细胞（EPCs）活化的关键因子。我们前期研究发现，侧支良好的冠心病患者外周血滤泡辅助性T细胞（Tfh）及其效应因子IL-21水平显著高于侧支不良者，此外小鼠后肢缺血组织中Nur77高表达同时伴LncRNA-Meg3表达降低。据此提出：激活的Tfh可能通过调控EPCs的TR3/Nur77-LncRNA-Meg3轴促血管新生的新机制。拟首先通过扩大样本的临床研究明确循环Tfh数量、活性与EPCs功能及侧支分级的相关性；随后体外研究初步揭示激活的Tfh调节EPCs的分子机制；最后分离Nur77-/-小鼠的EPCs，经预处理后与来自动脉粥样硬化模型小鼠的Tfh共培养，荧光标记后，将EPCs回输糖尿病后肢缺血小鼠，验证上述机制。本项目实施将为治疗性血管新生提供潜在靶点。

血管内皮炎症及功能异常与冠状动脉疾病密切相关，贯穿早期的斑块形成、斑块进展、微循环障碍及血管新生异常等多个环节，是冠心病的发生发展的重要机制。课题组推测孤儿核受体Nur77可以通过调节内皮细胞炎症而参与冠状动脉疾病发生发展及血管新生的调控。为此开展了评估冠心病患者外周血循环内皮祖细胞数量及活性、微循环功能及血管新生水平的临床研究，Nur77调控内皮细胞炎症及功能参与血管炎症性疾病进展及血管新生的机制研究等。结果发现：流式细胞分析提示冠心病患者人群中内皮祖细胞相对于健康对照人群显著降低（p<0.05），同时与侧枝循环密切相关，其中合并有糖尿病的冠心病患者水平尤为低下；进一步入选阻塞及非阻塞型阻塞型冠心病患者，采用激光散斑衬比分析成像技术及阻断后反应性充血法（PORH）行甲皱微循环检测评估外周微循环功能后发现非阻塞型冠心病患者外周微循环功能显著低于健康受试者及阻塞型冠心病患者（p<0.05）；随后在糖尿病后肢缺⾎模型小鼠中探讨了Nur77 对LncRNA-Meg3 表达的影响；并在实验室进行机制研究中发现，LPS可以刺激内皮细胞炎症小体中重要组分caspase-1表达与激活，进而促进IL-1β等炎症因子释放，从而导致HUVEC及微血管内皮细胞功能异常并引发血管炎症，而上调孤儿核受体Nur77的表达，可以显著抑制LPS诱导的caspase-1 的表达与激活，进而抑制细胞因子IL-1β的成熟与分泌，与之相对应的是，在Nur77基因敲除小鼠表现出血管炎症性疾病的显著加重，而给予caspase-1抑制剂的干预则明显减轻了炎症程度；进一步深入探讨发现，Nur77下调IRF-1的转录进而抑制IRF-1与 caspase-1启动子的结合是Nur77抑制血管内皮炎症的重要分子生物学机制。本课题首次证明了孤儿核受体Nur77可以通过抑制IRF-1/caspase-1介导的内皮细胞炎症而参与血管性疾病的调控。从一个崭新的方向揭示了血管炎症及内皮功能障碍的重要机制，为将来开发新型治疗方法提供理论依据。