PDE5基因修饰的脂肪干细胞移植治疗对大鼠肾脏缺血-再灌注损伤的保护作用及机制的研究

Adipose-derived (AD) MSCs are reported to promote regeneration of ischemia-reperfusion (IR) injury of the kidney because of the main advantages of minimal invasiveness in harvesting and unlimited supply from in vitro culturing. It has been shown that the long-acting phoshodiesterase5（PDE5） inhibitor, tadalafil, provides longer and sustained protection of mesenchymal stem cells and promotes their survival and proliferation in the infarcted heart.In this study, we'll test the hypothesis whether PDE-5 inhibition could improve the survival of ADSCs, which may lead to enhanced renal function following IRI in rat.We aim to observe whether PDE-5 inhibition by genetic knockdown with a silencing vector can improves survival following simulated IRI in vitro and in vivo transplantation of ADSCs by the role of cGMP-dependent PKG signaling pathway.What is the role of anti-inflammation, anti-apoptosis and paracrine mechanisms in enhancing cytoprotective effects of ADSCs with PDE-5 inhibition? Our study want to prove the hypothesis that PDE-5 inhibition is a powerful new preconditioning strategy to increase viability of ASCs in vitro and enhancing their therapeutic potential as shown by reducing inflammtion,apoptosis, improving vascular density,and renal function in rat following IRI.

近年来脂肪干细胞（ADSCs）应用于肾脏缺血再灌注损伤(IRI)治疗的研究已取得初步成果。现有研究发现抑制PDE同工酶5(PDE5)活性在体外可延长成体干细胞生存时间，同时可有效减轻实验动物器官缺血再灌注损伤。我们前期研究证实肾小管上皮细胞损伤（炎症、凋亡、转分化）与肾脏病理改变及肾功能损害密切相关。本课题通过沉默ADSCs中PDE5基因并建立稳定转染细胞株，观察其对ADSCs生存活力、增殖速度及旁分泌等性能的影响；并构建肾脏IRI的细胞及动物模型，通过肾小管上皮细胞分化、炎症、凋亡及旁分泌的研究探讨PDE5基因沉默的ADSCs移植治疗对缺血再灌注损伤的肾脏功能保护、肾脏组织修复的作用及其相关机制。本研究以期通过PDE5基因修饰优化脂肪干细胞效能，进一步增强ADSCs移植对缺血再灌注肾损伤的治疗作用，并深入探讨其可能的机制。

脂肪干细胞（ADSCs）应用于肾脏缺血再灌注损伤(IRI)治疗的研究已取得初步成果。现有研究发现抑制PDE同工酶5(PDE5)活性在体外可延长成体干细胞生存时间，同时可有效减轻实验动物心脏缺血再灌注损伤。我们通过构建沉默PDE5基因慢病毒包装系统转染大鼠ADSCs，筛选并成功建立稳定低表达PDE5基因的ADSCs细胞株(PDE5-shRNA ADSCs)；在体外缺血再灌注(I/R)细胞模型中，PDE5-shRNA ADSCs较正常细胞组增殖率显著增加，凋亡率明显下降，且上清液中VEGF、FGF水平及细胞内cGMP水平升高(均P＜0.05)，提示PDE5基因表达被抑制后可能通过上调cGMP水平，有效改善ADSCs在I/R状态下的增殖、凋亡及分泌功能。在大鼠肾小管上皮细胞体外I/R模型中，我们发现与PDE5⁃shRNA ADSCs 共培养的NRK⁃52E细胞较正常细胞组增殖显著增加，凋亡率明显下降，且HGF、FGF 水平升高(均P＜0.05)，且与阴性对照组比较，ADSCs上皮细胞标志物E⁃cadherin及CK18均表达显著增加(均P＜0.05)，提示PDE5⁃shRNA ADSCs通过促进干细胞分泌多种生长因子，有效改善肾小管细胞I/R状态下增殖及凋亡，同时促进自身向上皮细胞分化，为干细胞体内移植治疗肾脏缺血再灌注损伤中增加肾小管上皮细胞的生存活力、提升干细胞治疗效能提供了初步的实验基础。