Wnt信号通路在骨髓间充质干细胞修复急性肾损伤中的作用机制和靶向治疗研究

There still have been some challenges before mesenchymal stem cell (MSC) transplantation used in clinical treatment of acute kidney injury (AKI), such as limited homing rate, tumor formation and maldifferentiation, so it is greatly needed to find the direct mechanism of mesenchymal stem cell-mediated kidney repair in order to find convenient replacement drugs in clinical practice. It has been confirmed that the Wnt signaling pathway was involved in the whole process of kidney development and this vital pathway has been found to be reactivated in AKI recently. In the preliminary experiment, we found that MSC promote renal tubular epithelial cell regeneration after hypoxia/reoxygenation injury was associated with increased Wnt4 and β-catenin protein expressions. Thus we proposed that MSC would promote renal tubular regeneration after AKI through modulation of Wnt signaling pathway of injured tubular epithelial cells. In this project, we would use AKI models such as hypoxia/reoxygenation injury of HK-2 cell lines, three-dimensional chimeric renal tubular vesicles, and mice model of kidney ischemia/reperfusion injury, and then adopt microarray analysis, qRT - PCR, Western blot, gene transfection technology to screen and validate the importance of Wnt signaling pathway in the AKI repairing mechanism by MSC. Finally, we would test the possibility of new drugs targeted on Wnt pathway instead of MSC on AKI therapy in order to provide new ideas and new strategies for the prevention of AKI clinically.

间充质干细胞（MSC）移植用于临床急性肾损伤（AKI）治疗前尚存在一些问题，如归巢少、肿瘤形成和异位分化等，需要寻找MSC修复AKI的机制以便找到方便用于临床的替代药物。已证实Wnt信号通路参与胚肾发育的整个过程，且在AKI修复过程中也起重要作用。我们预实验发现，MSC促进低氧/复氧损伤肾小管上皮细胞再生修复过程与Wnt4和β-catenin蛋白表达增加有关，因此推测：MSC可能通过旁分泌作用激活受损肾小管上皮细胞Wnt信号通路，实现肾小管上皮细胞再生修复。本项目将以低氧/复氧损伤的HK-2细胞株、三维嵌合体肾小管囊泡和肾脏缺血/再灌注损伤小鼠为AKI模型，采用微阵列分析、qRT-PCR、Western blot、慢病毒载体转染等技术筛选和验证Wnt信号通路在MSC修复AKI中的重要作用，并进一步以Wnt通路为靶点寻找药物替代MSC治疗AKI的可行性，从而为AKI的防治提供新的思路和策略。

间充质干细胞（MSC）移植用于临床急性肾损伤（AKI）治疗前尚存在一些问题，如归巢少、肿瘤形成和异位分化等，需要寻找MSC修复AKI的机制以便找到方便用于临床的替代药物。已证实Wnt信号通路参与胚肾发育的整个过程，且在AKI修复过程中也起重要作用。本研究中我们旨在评估MSC条件培养基(CM)对顺铂诱导的AKI的保护作用和机制。在体外，我们选用鼠肾小管上皮细胞NRK-52E在有无CM的情况下与顺铂孵育，评估细胞活力、凋亡和细胞周期；ICG-001和IWR-1用于抑制wnt /β-catenin通路；使用DCFH-DA和MitoSOX评估细胞内和线粒体内ROS水平。在体内，大鼠造模成功后予静脉注射CM或MSC，第3天收集血清和肾脏组织，评估肾功能和组织学的变化。采用Western blotting 和qRT-PCR检测wnt /β-catenin通路相关基因和蛋白表达，采用免疫组化评估AKI患者肾组织内β-catenin的表达，采用Wnt激动剂BIO评估Wnt通路激活对AKI的治疗作用。我们发现，CM可以保护NRK-52E细胞免受顺铂损伤，其机制与激活wnt4/β-catenin通路有关。ICG-001和IWR-1可以逆转CM的保护作用，表现为细胞增殖减少，细胞凋亡增加，细胞内和线粒体ROS水平升高。采用siRNAs抑制β-catenin也可抑制线粒体保护因子PGC-1α、TFAM 和NRF-1。在大鼠模型中,CM显著缓解肾脏功能，减轻肾组织损伤，伴wnt4和β-catenin上调。临床资料显示,β-catenin高表达患者肾脏预后好于低表达患者。最后，采用Wnt激动剂BIO可通过抑制肾小管上皮细胞凋亡和线粒体氧化应激，促进肾小管上皮细胞在体内外的增殖，从而减轻顺铂诱导的急性肾损伤。因此我们认为,CM可以通过wnt4/β-catenin通路的激活预防顺铂导致的AKI,减轻细胞凋亡和细胞内ROS水平降低。Wnt激动剂BIO是有前景的防治顺铂肾损伤的治疗方法。