miR-92a在DCD供肾缺血/再灌注损伤中的作用及机制研究

Ischemia/reperfusion injury is classified as ischemia injury and reperfusion injury in cellular events, and both process are closely related to apoptosis. Kidneys from DCD donor suffered severe organ injury because of prolonged warm ischemia time, leading to further jeopardize to the organ function after reperfusion following transplantation. miR-92a is an inhibitor to many apoptosis related protein. In our previous study, we found elevated expression of miR-92a after renal ischemia/reperfusion injury, and enhanced miR-92a expression ameliorate the injury. That, we propose a hypothesis over expression of miR-92a can protect the DCD kidney from ischemia/reperfusion injury. To do that, we will collect samples from DCD kidneys and living donor kidneys, and analyze the relationship between the expression of miR-92a and tissue injury. Then, we will elevate or inhibit the expression of miR-92a in vivo and in vitro, to prove the over expression of miR-92a can ameliorate ischemia/reperfusion injury of DCD kidneys. Finally, we will interfere the expression of miR-92a at different steps, and investigate its impact on the quality of DCD kidneys and transplant outcomes. This research will provide a new point view to the ischemia/reperfusion injury of DCD kidneys and a new therapy to improve the quality of DCD kidneys.

缺血/再灌注损伤在细胞事件层面分为缺血损伤及再灌注损伤，均与细胞凋亡密切相关。较长时间热缺血及冷缺血会损伤心死亡(DCD)供肾，移植后再灌注进一步加重损伤，影响移植效果。miR-92a能够抑制多条凋亡通路的激活。在前期研究中，我们发现肾脏缺血/再灌注损伤后miR-92a明显增高，并证实其过表达能减少肾脏缺血/再灌注损伤。我们推测miR-92a过表达能够减少DCD供肾缺血/再灌注损伤。为证实这一假说，我们将收集DCD供肾组织及活体供肾组织，观察miR-92a的表达与缺血损伤及再灌注损伤的关系；再通过细胞模型及DCD供肾动物模型，观察干预miR-92a的表达对于DCD供肾缺血损伤及再灌注损伤的影响，并明确其信号传导通路；最后观察不同时机干预miR-92a的表达对于DCD供肾质量及移植后效果的影响。本课题将从新的视角阐明DCD供肾缺血/再灌注损伤发生的分子机制，并为改善DCD供肾质量提供新思路。

缺血/再灌注损伤在细胞事件层面分为缺血损伤与再灌注损伤，与细胞凋亡密切相关。较长时间热缺血及冷缺血会损伤心死亡(DCD)供肾，移植后再灌注进一步加重损伤，影响移植效果。在前期研究中，我们发现肾脏缺血/再灌注损伤(IRI)后miR-92a明显增高，并证实其过表达能减少肾脏缺血/再灌注损伤。采用动物及细胞模型均发现热缺血可以诱导自噬体形成；热缺血后冷保存及IRI明显增加自噬体数量，提示自噬水平明显增加；缺血后冷保存会明显降低miR-92a的表达，且随着时间延长，表达持续下降；而短时间再灌注会明显诱导miR-92a的表达，随着再灌注时间延长，miR-92a的表达持续下降。体外IRI模型也发现随着再富氧时间延长，miR-92a的表达持续下降。后续采用采用体外模拟IRI，我们发现miR-92a agomir 能够明显减少缺氧及缺氧再富氧所诱导的HK-2细胞凋亡，而miR-92a antagomir能够明显触及缺氧及缺氧再富氧所诱导的细胞凋亡。采用自噬促进剂雷帕霉素(Rapa)能够明显促进缺氧再富氧所诱导的凋亡，miR-92a能够拮抗Rapa的促凋亡作用。进一步我们还发现miR-92a agomir能够明显抑制缺氧及缺氧再富氧所诱导的自噬水平升高，也能够拮抗rapa所诱导的自噬水平增加。进一步我们发现miR-92a是通过MEK/JKN通路抑制HK-2细胞缺氧再富氧条件下的自噬水平，从而抑制凋亡。这些结果证过表达miR-92a，可以改善肾脏保存质量及减少缺血再灌注损伤。