人尿源性干细胞通过外泌体传递miR-216a-5p减轻肾缺血-再灌注损伤的作用及机制研究

Renal ischemia-reperfusion (I/R) injury plays a major role in acute kidney injury (AKI). Due to less knowledge of the repairing mechanism after renal I/R injury, its treatment is limited, and the mortality rate is high. Our previous studies have found that exosomes secreted from human urine-derived stem cells (USCs) can reduce renal I/R injury in rats; and co-culturing them with renal tubular epithelial cells exposed to hypoxia reoxygenation (H/R) can decrease the apoptosis, accompanied with the down-regulation of PTEN and activation of Akt phosphorylation. Besides, miR-216a-5p was found notably high in exosomes from human USCs. Therefore, based on the bioinformatic prediction and our previous studies we hypothesize that the exosomes from human USCs transfer miR-216a-5p to reduce renal I/R injury by the inhibition of apoptosis through regulation of PTEN/Akt pathway. In this study, we will employ the H/R cell model to investigate if miR-216a-5p in exosomes from USCs can be taken up by renal tubular epithelial cells and reduce their apoptosis through regulating the PTEN/Akt pathway. Furthermore, we will use renal I/R injury rat model to explore the expression of miR-216a-5p in the injured kidney tissues and validate if it can improve the impaired renal function. This study will help to further elucidate the repairing mechanism of renal I/R injury and provide new ideas for the treatment and prevention of AKI.

肾缺血-再灌注（I/R）损伤是急性肾损伤（AKI）的重要原因；因肾I/R损伤的修复机制不清，所以治疗手段有限，死亡率高。申请人前期研究发现，人尿源性干细胞（USCs）分泌的外泌体可减轻大鼠肾I/R损伤，将其与缺氧-复氧（H/R）条件下的肾小管上皮细胞共培养，发现细胞凋亡减少，PTEN降低，Akt磷酸化增强；且miR-216a-5p在USCs外泌体中表达显著升高。基于生物信息学预测和前期工作基础，本项目研究假说是：USCs的外泌体通过传递miR-216a-5p调控PTEN/Akt通路，抑制凋亡，减轻肾I/R损伤。本项目将利用H/R细胞模型研究携带miR-216a-5p的USCs外泌体被肾小管上皮细胞摄取，并作用于PTEN/Akt通路，减少细胞凋亡的机制；再利用肾I/R损伤大鼠模型验证其在肾脏的分布及对肾功能的保护作用。本项目将有助于阐明肾I/R损伤修复机制，为AKI的临床防治提供新思路。

项目背景：缺血-再灌注（ischemia/reperfusion，I/R）损伤是急性肾损伤（acute kidney injury，AKI）的重要机制之一，在临床上常见于各种休克、心肌梗死、移植以及手术时临时血管阻断等。因肾脏 I/R 损伤的修复机制不清，所以治疗手段有限，死亡率高。有研究报道人尿源性干细胞（urine-derived stem cells，USCs）可以减轻大鼠缺血性肾损伤，但机制不明。.主要研究内容：（1）USCs的体外培养和鉴定以及其分泌的外泌体的提取和鉴定；（2）通过体外缺氧-复氧（hypoxia/reoxygenation，H/R）的细胞实验研究USCs和其来源的外泌体对肾小管上皮细胞HK-2 H/R损伤的作用；（3）对尿源性干细胞外泌体中miRNA富集分析，筛选差异表达的miRNA，通过过表达和敲减miRNA研究其在HK-2细胞H/R损伤中的作用和相关的分子调控机制；（4）利用I/R大鼠模型进行体内实验，验证USCs来源的外泌体和其中富集的miRNA在改善肾脏功能、减轻细胞凋亡中的作用和分子机制。.重要结果和关键数据：（1）成功分离培养了人USCs，提取了其分泌的外泌体，并对外泌体的形态、粒径和特异性标志物进行了鉴定；（2）体外细胞实验结果证实USCs来源的外泌体在HK-2细胞的H/R损伤中发挥保护作用；（3）USCs外泌体中富含miR-216a-5p，可以靶向调控PTEN的表达，通过Akt通路影响细胞凋亡。在H/R条件下的HK-2细胞与USCs来源的外泌体共孵育，或用miR-216a-5p类似物均可以使HK-2细胞中miR-216a-5p上调，PTEN下调，Akt磷酸化水平增高，反之，用anti-miR-216a-5p转染的USCs中的外泌体可以有效抑制H/R损伤的HK-2细胞中miR-216a-5p的水平，同时抑制Akt磷酸化，恢复PTEN表达，并促进细胞凋亡。双荧光素酶实验证实了miR-216a-5p和PTEN的相互作用。（4）大鼠肾脏I/R模型进一步证实了静脉给予USCs分泌的外泌体和miR-216a-5p可以有效改善损伤的肾脏功能，减轻病理损伤和细胞凋亡，且USCs来源的外泌体可以抑制大鼠肾脏PTEN的表达。.科学意义：本项目有助于阐明肾I/R损伤的修复机制，为临床寻找有效的AKI保护干预措施和治疗靶点提供新思路。