骨髓间充质干细胞调节炎性微环境干预恒河猴糖尿病肾病免疫损伤的效应机制

Inflammatory response is a key factor in the development of diabetic nephropathy (DN). Mesenchymal stem cell(MSC) has been shown could repaie of renal injury and reverse the progress of DN in rodents. Our research group has established DN model of rhesus monkeys.Thus, to explore the mechanism of MSC for intervention of rhesus monkeys with early diabetic nephropathy has important scientific and practical significance. The project is proposed on the following basis: 1) rhesus monkey model of early DN induced by high fat and sugar; 2) study the repairing mechanisms of MSC on oxidative stress injury of endothelial cell in vitro. After that, separated and amplified MSC from monkeys with DN, labeled with superparamagnetic iron oxide （SPIO）, and multiple reinfused of autologous MSC in the manner of percutaneous renal arterial interventions. The subjects were evaluated by the following parameters in the different course of disease: 1) blood biochemistry and urine routine; 2) analyzed the migration and homing law of stem cells in vivo with 7T Magnetic Resonance Imaging; 3) Color Doppler ultrasound analyzed of renal hemodynamic parameters; 4) flow cytometer analyzed the distribution of lymphocyte subsets; 5) renal biopsy observed lesion of microenvironment, expression of inflammatory genes, and the effect of tissue repairing. Finally, we researched the correlation between parameters in the different course of disease, and investigated the mechanisms of MSC on the effect of modulating the immune system, repairing oxidative stress injury of endothelial cell, and reversing renal injury. This study will help to elucidate the mechanism of MSC on the effect of regulating inflammation and repairing microvascular, as well as provide safe and effective stem cell therapy program for DN treatment.

炎性反应是糖尿病肾病(DN)发展的关键因素，MSC在小鼠体内有修复肾损伤的现象。利用MSC的免疫调节功能和课题组已建立的恒河猴DN模型，探索MSC调节微环境炎性反应逆转DN免疫损伤的效应机制具有重要科学价值和实践意义。 本项目拟建立血管内皮细胞高糖、缺氧和炎性应激损伤模型，研究MSC对该损伤干预的分子机制；在此基础上，将SPIO标记的恒河猴MSC经皮穿刺肾动脉，介入方式回输恒河猴早期DN模型肾内；于不同治疗时相观察恒河猴血/尿生理指标、7T磁共振分析MSC体内迁移及归巢规律、彩超分析肾血流动力学改变、流式分析淋巴细胞亚群分布、穿刺肾脏观察病变组织微环境的炎性分子表达谱和损伤修复效果；分析DN体液、影像和组织炎性分子改变的规律及其相互关系，验证MSC调节免疫、修复血管内皮应激和逆转肾损伤的机制和效果，阐明MSC在DN炎性调节和微血管修复中的作用机理，为DN治疗提供安全有效的干细胞治疗方案。

本项目按计划执行，取得以下研究成果：.1.建立了恒河猴糖尿病肾病(DN)模型：糖尿病建模2年后恒河猴出现肾脏的早期损伤，而钠和脂肪摄入可加重肾脏病变。血尿生化、肾脏穿刺的组织学及纤维化蛋白检测和肾脏超声造影技术可以在不同时期评价糖尿病肾损伤的程度；肾脏致纤维化因子表达和病理改变早于微量蛋白尿和肾功能改变。.2.MSCs输注治疗恒河猴DN：共聚焦显微镜观察发现CM-DiL标记MSCs多定位于免疫器官，也可归巢肾脏且以肾小管周为多。MSCs治疗3月后能改善血液肾功能指标和脂代谢紊乱；减少尿样微量白蛋白以及UACR；肾穿刺发现病理改变减轻，ECM蛋白沉积减少，炎症和趋化因子水平下降；电镜观察显示：肾小球基底膜和肾小管EMT的程度都有所缓解。为MSCs用于临床DN治疗提供了可靠的灵长类动物实验证据。.3.MSCs改善糖尿病肾脏炎性微环境缓解肾损伤的机制研究.1）改善Mφ极化和炎症微环境.*MSCs可提高DN模型M2型Mφ，抑制M1型Mφ的分化，改善Mφ中溶酶体-自噬紊乱，抑制高糖诱导的Mφ炎性分子表达，有效抑制DN鼠肾脏的炎症反应，改善肾功能。.*体外研究发现MSCs通过激活Mφ线粒体生物合成、抑制Cyt-C的释放，促进线粒体能量代谢，修复高糖诱导的Mφ线粒体损伤、提高溶酶体-自噬过程，保护肾小管上皮细胞功能。.2）对血管内皮细胞损伤的干预.*MSCs抑制高糖诱导的线粒体ROS累积，通过激活SIRT1/AMPK/PGC-1α 通路促进内皮细胞的线粒体生物合成，缓解血管内皮的氧化损伤。.*针对高脂损伤，MSCs能通过分泌的TSG-6提高内皮的增殖活性、减少凋亡、抑制炎性因子和NF-κB活化，改善内皮细胞功能。.*MSCs可减少TNF-α诱导的细胞内ROS积累，其分泌的STC-1能通过上调Hp-AMPK，促进UCP-2的表达；UCP-2可通过解耦联作用降低细胞内ROS积累，从而达到缓解内皮炎性损伤。.3）对肾小管细胞损伤的干预.*MSCs微泡内高表达miR-451a，miR-451a可下调小管细胞P15、P19的表达重启受阻周期；抑制高糖高尿酸诱导的EMT过程，改善肾功能。临床DN患者miR-451a和P19水平与病程相关。.*MSCs能通过分泌IL-6改善高糖和炎性因子诱导的HK2炎症和纤维化因子表达，抑制NF-κB活化；还能改善HK2的葡萄糖重吸收功能，缓解EMT。