骨髓间充质干细胞通过外泌体LncARSR调控YAP蛋白促进血管新生改善严重下肢缺血的机制研究

Critical limb ischemia is a serious disease caused by atherosclerosis. The key to treatment is to correct and reverse the vascular endothelial injury. We found in previous studies that LncARSR-overexpressing mesenchymal stem cells (MSCs) can promote the proliferation of vascular endothelial cells when co-cultured. Further studies showed that LncARSR was also up-regulated in exosomes derived from this MSCs. YAP expression is increased in co-cultured vascular endothelial cells. This suggests that MSCs may transmit LncARSR into vascular endothelial cells through the exosome pathway and increase YAP expression to promote angiogenesis and repair. In order to further explore its mechanism, this project intends to carry out the following studies: (1) to study the changes of genetically modified exosomes by molecular biology to investigate the regulatory effect of LncARSR and YAP protein binding sites and LncARSR on YAP protein expression ; ② intervention of YAP protein expression to verify the role of YAP promote angiogenesis; ③ in vitro and in vivo validation of genetically modified LncARSR-YAP axis MSCs exosomes on vascular endothelial injury treatment. To find a new treatment of critical limb ischemia provides a theoretical basis and direction.

严重下肢缺血是动脉粥样硬化导致的严重病变，治疗的关键在于纠正和逆转血管内皮损伤。我们在前期研究中发现过表达LncARSR的间充质干细胞（MSC），可促进与其共培养的血管内皮细胞增殖；进一步研究发现，该MSC来源的外泌体中LncARSR也表达上调；同时，发现与其共培养的血管内皮细胞中YAP表达增加。这提示MSC可能通过外泌体途径传递LncARSR至血管内皮细胞中，增加YAP表达以促进血管再生修复。为进一步探索其机制，本项目拟开展以下研究：①通过分子生物学方法研究基因修饰后外泌体的变化水平，探究LncARSR与YAP蛋白的结合位点和LncARSR的水平对YAP蛋白表达的调控作用；②干预YAP蛋白的表达，验证YAP促进血管新生的作用位点；③在体内外水平验证基因修饰LncARSR-YAP轴通过MSCs外泌体对血管内皮损伤的治疗效果。为寻找严重下肢缺血新的治疗手段提供理论依据和方向。

严重下肢缺血（CLI）是动脉粥样硬化导致的严重病变，治疗的关键在于纠正和逆转血管内皮损伤。我们在前期研究中发现过表达LncARSR的间充质干细胞（MSC），可促进与其共培养的血管内皮细胞增殖；进一步研究发现，该MSC来源的外泌体中LncARSR也表达上调；同时，发现与其共培养的血管内皮细胞中YAP表达增加。这提示MSC可能通过外泌体途径传递LncARSR至血管内皮细胞中，增加YAP表达以促进血管再生修复。本项目通过RNA Pull-down，Co-IP等分子生物学方法证明过表达LncARSR可以提高MSC产生的外泌体LncARSR含量，且证明LncARSR与YAP存在结合位点，可以稳定YAP蛋白的表达；LncARSR与YAP均可以促进内皮细胞增殖，且可以促进内皮细胞中血管生成素2表达；在细胞及动物水平验证过表达LncARSR增强MSC通过LncARSR-YAP轴增强对CLI的治疗作用。本研究为寻找CLI新的治疗手段提供了理论依据及方向。