PEG修饰的人脐带血间充质干细胞外泌体对铀中毒的救治作用及机制研究

In recent years, depleted uranium weapons are widely used in the war. Depleted uranium waste generated by the nuclear industry is gradually increasing, which is harmful to the environment and people's health. The key to the prevention and treatment of depleted uranium is to find out the ideal drug for decorporation. However, it has been proved that the delayed delivery of the drug is not effective, and the long-term administration of the drug has a large renal toxicity. Mesenchymal stem cells (MSCs) can reduce the mortality and protect kidney injury, has the strongest ability to produce exosomes(EXs). The EXs from MSCs has a better therapeutic effect on multiple organ damage, including the promotion of survival and regeneration in vitro and in vivo. But exogenous EXs lack of specific targeting, and can be rapidly cleared from the circulation, the protection effect is limited. In this study, we designed EXs, targeting ligand and polyethylene glycol (PEG) to connect the epithelial growth factor receptor (EGFR) nanoparticles to phospholipid (DMPE) -PEG, forming the nano-PEG micelles. Then compared with sodium bicarbonate, MSC, MSC-EXs control group, the protection effect of the nano-PEG micelles on uranium poisoning is investigated. Molecular biology, proteomics and other methods were used to study the mechanism of accumulation, metabolism and related signals, to find specific target.

近年，贫铀武器广泛用于战争，核工业产生的贫铀废料逐渐增多，污染食物和水源,对环境和居民健康造成一定危害。防治贫铀损伤的关键是找出理想的促排药。但已有促排药延迟给药效果不佳，长期给药，蓄积致肾毒性大。间充质干细胞（MSCs）可降低肾毒性动物的死亡率，保护肾脏损伤，且产生外泌体（EXs）能力最强。来源MSCs的EX对多种器官损伤疗效更好，包括对离体和在体肾脏具有促进存活和明显再生作用，易分离，更安全，无肝肾毒副反应。但外源性EXs缺乏特异靶向性，且可迅速从循环中清除，疗效受限，故本研究设计了将EXs、靶向配体与聚乙二醇（PEG）连接，将上皮生长因子受体（EGFR）纳米体连接到磷脂（DMPE）-PEG，形成纳米-PEG胶束；将其与促排剂碳酸氢钠、单纯MSC、MSC-EXs等对照，研究对铀中毒的救治效果；使用分子生物学、蛋白组学等方法，从体内蓄积、代谢和信号通路等途径研究其机制，为找到靶点打下基础

传统的铀中毒治疗方法偏向于对使用化合物与铀酰离子络合，形成水溶性的化合物，促进其排出，其副作用较大，且残留在体内的贫铀放射性是持久的。因此，铀中毒的治疗，不仅要促进铀的促排，还要修复受损伤的组织，才能达到治疗目的。本研究设计了将EX、靶向配体与聚乙二醇（PEG）连接，将上皮生长因子受体（EGFR）纳米体连接到磷脂（DMPE）-PEG，形成纳米-PEG胶束；通过动物水平研究了人脐带MSC-EXs对铀中毒的救治效果，发现外泌体能够改善铀中毒小鼠肾功能指标，改善肾脏和骨髓的病理变化，降低血液、肾脏和骨髓U含量，促进尿液U排出；细胞水平发现，提前给予外泌体可保护HK-2细胞免受贫铀伤害，降低细胞内ROS和MDA水平，提高GSH水平，增强SOD活力，降低细胞的凋亡。其机制是一方面，MSC-EXs可能通过靶向受损伤的组织器官，使与铀酰离子结合的蛋白如白蛋白、乳铁蛋白和骨桥蛋白等的表达下调，降低铀蓄积器官的损伤，降低血液、肾脏和骨骼等组织铀含量，减轻肾脏的炎症损伤。另一方面，MSC-EXs可减轻细胞ROS的产生，通过上调HSP等的表达，减轻铀引起的氧化应激；稳定线粒体膜电位，抑制线粒体途径凋亡的激活，通过抑制促凋亡蛋白Bax的表达，促进抑凋亡因子Bcl-2的表达；抑制同源活化、启动凋亡的Caspase9表达，进一步抑制线粒体途径的细胞凋亡，也继之抑制异源活化、将凋亡信号放大、将死亡信号向下传递的Caspase3的表达，减轻细胞凋亡实现。这些结果表明，MSC-EXs具有成为新的铀中毒治疗剂的潜力。