端粒保护蛋白复合体POT1-TPP1调控内皮祖细胞促进支架内皮化进程的作用及机制研究

Late stent thrombus (LST) can cause acute myocardial infarction, or even sudden death. However, the exact mechanism of LST has not been clarified yet. Incomplete stent endothelialization resulting from endothelial progenitor cell (EPC) senescence is considered as one of the most important mechanism related to LST. Our group has reported for the first time that leukocyte telomere length is inversely associated with the percent stent strut coverage, but the mechanism is unclear. Previously studies have demonstrated that telomere length which is closely related to cell senescence determines the age of cells. Moreover, Inflammation is one of the most important injury factors thus lead to telomere shortening. While latest researches have found that POT1-TPP1 telomere protein complex which binding to the telomere DNA terminus can maintain normal structure and function of telomere as a result of protecting telomere from shortening and being damaged. Our previous animal study has showed that there is a close relationship with stent endothelialization delay and inflammation. Therefore, we deduce that local and systematic inflammation after stent implantation can alter the expression of POT1-TPP1 protein complex which ultimately accelerate telomere attrition and EPC senescence. This may be a new mechanism of LST. To validate our hypothesis, we investigate the effect of POT1-TPP1 on telomere length, as well as the proliferation and differentiation of EPC and pathway involved by alteration of POT1-TPP1 level using techniques such as gene transfer, flow cytometry and so on. This study will clarify a new molecular mechanism of LST and thus provide a new target for prevention and control.

晚期支架内血栓(LST)可导致心肌梗死甚至猝死。LST 确切机制尚未阐明，多认为与内皮祖细胞（EPC）老化所致的支架内皮化不全有关。本课题组首次证实白细胞端粒长度的缩短与支架内皮覆盖不全有关。研究显示端粒的长度与细胞衰老密切相关，且炎症是造成其损伤和缩短的最主要因素之一。新近研究发现端粒结构和功能的维持依赖于保护复合体POT1-TPP1。前期动物研究我们发现支架内皮化延迟与炎症反应密切相关。因此，我们推测支架后局部和系统性炎症影响了POT1-TPP1的表达和功能，导致端粒缩短和EPC老化加速。这可能是LST的一个新机制。为证实这一假说，我们应用基因转染、流式细胞仪等技术，炎症刺激下观察EPC老化以及POT1-TPP1的表达，从整体和细胞两个层次，观察该复合体对EPC的衰老，增殖分化功能，支架内皮化的影响及机制。本课题将从新的视角阐明 LST发生的分子机制，并为 LST的防治提供新靶点。

内皮祖细胞（EPC）老化不仅参与动脉粥样硬化斑块的形成与进展，而且在支架内皮化进程中扮演着至关重要的角色。而支架内皮化不全与晚期支架内血栓的形成密切相关，此外内皮祖细胞的老化严重影响血管内皮细胞损伤修复的功能，导致内皮细胞功能下降甚至过早凋亡，从而引起冠脉内急性血栓的形成(斑块侵蚀)，导致心肌梗死的发生。本课题组首次证实白细胞端粒长度的缩短与支架内皮覆盖不全有关。既往研究显示端粒的长度与细胞衰老密切相关，且炎症是造成其损伤和缩短的最主要因素之一。新近研究发现端粒结构和功能的维持依赖于保护复合体 POT1-TPP1。我们的动物研究证实支架内皮化延迟与炎症反应密切相关。因此，在本课题的支持下，经过三年的时间，我们应用基因转染、流式细胞仪等技术，炎症刺激下观察 EPC 老化以及 POT1-TPP1 的表达，从不同层次，观察该复合体对 EPC 的衰老，增殖分化功能。通过体外实验，我们证实了炎症反应通过诱发端粒的关键保护蛋白TPP1的水平下降，进而引起内皮祖细胞功能减退和老化。而上调端粒结合蛋白TPP1表达水平，可有效改善内皮祖细胞的功能，延缓内皮祖衰老。由于研究中遇到难以克服的难题，同时为了使本项目的研究更具临床指导价值，我们在对实验方案进行调整后，增加了临床试验部分。前瞻性入选65例急性冠脉综合征（ACS）患者，所有患者于支架植入前和术后即刻以及术后6个月均行高分辨率光学相干断层成像（OCT）扫描，评价罪犯病变斑块特征和术后即刻血管损伤并发症以及支架内皮化程度。研究结果显示患者外周血内皮祖细胞的TPP1水平与OCT检测的支架内膜覆盖有很好的相关性，此外，内皮祖细胞端粒酶活性与外周血高敏C反应蛋白水平成负相关，进一步验证我们的前期假设。. 在本项目的支持下，课题组已发表相关文章5篇，培养硕士研究生2名，博士研究生1名。