体外循环前内皮祖细胞预动员启动肾血管内皮细胞修复机制研究

Acute kidney injury is one of the major complications after cardiopulmonary bypass (CPB). Currently, vascular endothelial cell injury induced by CPB is taken as one of the main causes related with kidney injury, and the "best time" for vascular endothium repairment may be the first 24 hours after injury. Our pilot studies have shown that circulating endothelial progenitor cell (EPC) may play an important role in repairing renal vascular endothelium via selectively homing to kidney. Unfortuately, we also found that the counts of EPC, as well as EPC stimulator levels, were significantly decreased after CPB, suggesting the repairing of renal vascular endothelium most depends on EPC produced before CPB. We, therefore, hypothesized that increasing EPC counts preoperatively (which we named pre-mobilization) would enhance renal vascular endothelium repairing and thereafter improve renal function. Based on these results, in the project, EPC mobilization via Granulocyte Colony Stimulating Factor (G-CSF) will be observed, and the function of the mobilized EPC will be tested in 3D cell culture system. Then the protective effect of pre-mobilized EPC on kidney will be observed in the established novel mouse CPB model. Using EPC VEGF/HGF gene silencing, GFP-mouse and myeloablative mouse model, the mechanisms whereby will also be elucidated to determine whether the protective effects were through EPC direct differentiation and/or paracine cytokines to urgently repair the renal blood vessel. Obviously, the present project will provide novel theoretical and experimental bases to prevent and treat acute kidney injury associated with CPB, and thus improve the outcomes after CPB for the short terms and the long terms.

急性肾损伤是体外循环（CPB）后主要并发症及死亡原因，与CPB引起的血管内皮细胞损伤密切相关，而损伤后24小时内是修复内皮细胞、减轻肾损伤的"最佳时间"。我们的前期研究发现，循环内皮祖细胞（EPC）可能对肾血管修复起重要作用，然而CPB后24小时EPC及其动员因子显著下降，提示这种修复主要依赖于术前产生的EPC。因此如在CPB前增加EPC（我们称为预动员）可能通过促进肾血管修复而改善肾功能。为此本项目拟研究粒细胞集落刺激因子对EPC预动员的时效关系；使用三维立体培养模型，研究预动员对EPC功能的影响；利用业已建立的小鼠CPB模型，研究预动员EPC对肾脏的保护作用；利用GFP-荧光小鼠、清髓小鼠模型，以及基因沉默技术，研究EPC是否通过直接分化作用和/或旁分泌作用而紧急修复肾血管。本课题的顺利实施为临床预防和治疗急性肾损伤，从而改善CPB后患者预后提供新的理论和实验依据。

研究背景：体外循环(Extracorporeal Circulation, ECC)是心脏直视手术不可或缺的辅助及治疗手段。ECC相关的全身炎症反应（Systemic Inflammatory Response, SIR）及多器官损伤仍然是难以回避的并发症。急性肾损伤(Acute Kidney Injury, AKI)是ECC术后常见的并发症之一，不仅增加住院日期和住院费用，还明显增加术后患者的死亡率，极大地威胁着临床患者的预后。近年来，以干细胞为基础的预防及治疗方案受到越来越多的重视。CD133+祖细胞一类具有多种用途的干细胞，主要存在于骨髓等器官，循环中也有少量的存在。研究报道，在药物诱导的AKI小鼠模型，CD133+祖细胞可以减轻肾损伤，改善肾功能。然而，CD133+祖细胞在ECC相关AKI的作用尚不清楚。.研究目的：本研究旨在探索CD133+祖细胞对ECC相关AKI是否具有保护作用，并初步探讨可能的机制，为临床应用提供理论和实验基础。.主要研究内容：研究通过设计三部分实验，首先，通过观察临床上行心脏瓣膜置换术的风湿性心脏病患者循环CD133+祖细胞数量与血清酐、尿素氮水平，初步探索循环CD133+祖细胞是否对ECC术后AKI有保护作用；其次，建立小鼠动静脉部分转流（Arterial-Venous Partial Bypass, AVPB）相关AKI模型，模拟临床ECC，诱导急性肾损伤；最后，在小鼠AVPB相关AKI模型的基础上，通过“预动员”的方式提高术前循环CD133+祖细胞数量，探讨“预动员”CD133+祖细胞在ECC相关AKI中的肾保护作用及机制。.研究结果：实验一证实了循环CD133+祖细胞在ECC相关AKI中具有肾保护作用。实验二成功建立了小鼠AVPB相关AKI模型，能够复制ECC过程，诱发AKI。实验三证实了“预动员”CD133+祖细胞可通过旁分泌机制，调控肾脏炎性反应，减轻ECC相关AKI，改善肾功能。.结论：“预动员”的CD133+祖细胞可在ECC开始后，迅速归巢至受损的肾小球区域，通过旁分泌机制减轻肾组织炎症反应，减轻肾损伤，改善肾功能。这为临床上治疗/预防ECC相关AKI提供了理论与实验基础。