外泌体作为circRNA的新型载体清除衰老细胞并延缓心脏衰老的策略及机制研究

Aging is a major cause of mortality and morbidity. So far, there is no effective treatment to delay the aging process. Recently, a new strategy aimed at delaying aging through the removal of senescent cells has received much attention from the scientific community. However, this strategy has not been used to prevent aging in the heart. We found that exosomes derived from umbilical mesenchymal stem cells can restore cardiac function in old mice, and the beneficial effect was mediated through the inhibition of senescent pathway by exosome-derived circular RNAs. In this grant application, we propose the following studies: 1) To delineate the mechanisms by which exosome-derived circular RNAs remove senescent cells and delay aging process using small-interfering RNA-based gene silencing and lentivirus-based gene overexpression, and to identify new target for the prevention of aging at early stage; 2) To prolong exosome retention in hearts by encapsulating the exosomes in nanomaterials protecting the hearts; 3) To construct cardiac specific circular RNAs knockout and overexpression mice in order to provide a new model for anti- aging research, and to further define the mechanisms whereby exosome-derived circular RNAs remove senescent cells and delay aging process. The goal of this study is to provide both an experimental and a theoretical basis for stem cell-derived exosomes as novel circular RNAs carriers to remove senescent cells and to delay aging in the heart, and to provide new strategy for the intervention of aging process.

衰老是人类致残致死的主要原因，目前尚无有效方法来减缓衰老进程。近期，通过清除衰老细胞从而延缓衰老的新策略备受研究者关注，但均不是针对心脏。我们先前研究结果发现，间充质干细胞来源的外泌体能有效地恢复年老小鼠的心功能，并且这种对衰老相关通路的抑制作用是由外泌体内circRNA介导的。基于此，本研究的内容主要包括以下几点：1）用siRNA抑制、慢病毒系统过表达等多重策略，阐明外泌体中circRNA清除心脏衰老细胞并延缓衰老的具体机制，筛选出早期预防心脏衰老的靶点；2）使用具有心脏保护作用的纳米材料包封外泌体，提高其在心脏的驻留率；3）构建心脏特异性敲除和过表达circRNA小鼠，提供研究衰老的新模型，并在体内进一步验证外泌体中circRNA延缓衰老的机制。本研究旨在为干细胞来源的外泌体作为circRNA新型载体清除衰老细胞，并延缓心脏衰老提供实验数据和理论基础，为延缓衰老提供新方法。

衰老会诱导心血管系统结构和功能的改变，同时也会诱导缺血性疾病的发生，导致病死率升高，但相关机制并不完善，也尚无有效方法来减缓衰老进程。对此，本项目的主要研究内容包括：1）用circRNA/mRNA测序、蛋白质测序、质谱分析、circRNA和蛋白质的免疫共沉淀、蛋白质之间的免疫共沉淀、核质分离、siRNA抑制剂、慢病毒过表达系统、分子信息学等多重策略，筛选出预防衰老的靶点，阐明外泌体中circRNA抑制心脏衰老、修复缺血组织损伤的具体机制；2）使用具有心脏保护作用的纳米多肽包封间充质干细胞来源的外泌体(UMSC-Exos)，提高其在心脏的驻留率；3）构建心肌细胞特异性敲除circHIPK3小鼠，提供研究衰老的新模型，并在体内、体外进一步验证UMSC-Exos中circHIPK3延缓衰老的机制。项目研究发现，1）UMSC-Exos可作为一种良好的生物载体递送circHIPK3至缺血损伤组织区域，靶向调控miR-421/FOXO3a表达，减少IL-1β和IL-18的释放，抑制由NLRP3/Caspase-1/IL-1β信号通路激活诱发的炎症小体反应及焦亡，修复缺血损伤；2）纳米多肽PGN水凝胶能够有效地包封UMSC-Exos并确保UMSC-Exos的稳定性和持续释放，改善心脏功能；3）团队成功构建了心肌细胞特异性circHIPK3基因敲除小鼠，并发现circHIPK3的缺失导致心肌细胞衰老和心脏功能下降。UMSC-Exos通过传递circHIPK3可显著发挥心脏保护作用。我们创新性发现，circHIPK3作为分子胶增强了细胞质中E3泛素连接酶β-TrCP和HuR的结合，导致p21mRNA结合蛋白HuR的泛素化降解，降低了衰老诱导因子p21的表达及活化，延缓了心脏衰老。综上，本项目提出了circHIPK3可作为分子胶，加速p21降解的新理论，这不仅是circRNA领域的一个新理论（以前主要认为circRNA是miRNA、mRNA的海绵），也为衰老及衰老引起的心血管疾病、缺血性疾病研究提供了实验数据和理论基础，为延缓衰老提供了新策略。