间充质干细胞外泌体调控氧化应激减轻D-氨基半乳糖/脂多糖诱导的急性肝损伤

Regulation of oxidative stress by exosomes derived from mesenchymal stem cells protects against D-galactosamine hydrochloride/lipopolysaccharides induced acute liver injury

目的:探讨小鼠间充质干细胞来源的外泌体(exosome,Exo)对D-氨基半乳糖(D-galactosamine hydrochloride,D-GalN)/脂多糖(lipopolysaccharides,LPS)诱导的急性肝损伤治疗作用及可能机制。方法:提取小鼠间充质干细胞,通过流式细胞术和细胞分化实验进行验证。超速离心法提取间充质干细胞外泌体,透射电镜和蛋白质免疫印迹对其鉴定,纳米颗粒追踪分析测定外泌体粒径电位。通过活性氧荧光探针考察外泌体减轻活性氧损伤的功能。CCK-8检测外泌体对肝细胞的保护作用。腹腔注射D-GalN/LPS制备急性肝损伤小鼠模型,考察外泌体对D-GalN/LPS诱导的急性肝损伤小鼠的治疗作用。结果:原代间充质干细胞表达CD29和CD44两个标志蛋白,而不表达CD31和CD117,具有成骨分化能力。间充质干细胞外泌体为具有典型杯状结构的纳米囊泡,表达CD9和CD63两个标志性蛋白。与PBS组相比,干细胞外泌体能够减轻L02细胞内的活性氧水平,提高L02细胞活性,且外泌体本身对L02细胞活性没有影响。与PBS组相比,Exo治疗组能够改善肝脏组织损伤,降低转氨酶水平,调节丙二醛、过氧化氢酶和超氧化物歧化酶的水平。结论:在细胞实验中,间充质干细胞外泌体能够减少活性氧,保护肝细胞的活性;在动物实验中,间充质干细胞外泌体能够下调氧化应激相关蛋白的表达,促进肝脏组织修复。

Objective:To explore the therapeutic effect and possible mechanism of exosomes derived from mouse mesenchymal stem cells(MSC)on D-galactosamine hydrochloride/lipopolysaccharides induced acute liver injury.Methods:Mouse mesenchymal stem cells were extracted and detected by flow cytometry and cell differentiation experiment.The exosomes of MSC were collected and purified by ultracentrifugation and identified by transmission electron microscopy and Western blotting.Size distribution and zeta potential of exosomes were measured by NTA.The ability of exosomes to reduce the oxidative damage was detected by reactive oxygen species(ROS)fluorescence probe.CCK-8 assay was used to investigate the protective effect of exosomes on hepatocytes.The therapeutic effect of exosomes on D-GalN/LPS-induced ALI was also evaluated.Results:Primary mesenchymal stem cells expressed CD29 and CD44 but did not express CD31 and CD117.The exosomes derived from mesenchymal stem cells were nano-sized vesicles with typical cup-shaped structure,which expressed CD9 and CD63.Compared with PBS group,exosomes could reduce the level of ROS and improve the viability of L02 cells,while exosomes themselves had no obvious effect on cell viability.Compared with PBS group,exosomes could reduce liver damage and transaminase level,regulate the levels of malondialdehyde,catalase and superoxide dismutase.Conclusion:At the cellular level,exosomes derived from mesenchymal stem cells could reduce ROS level and protect hepatocyte viability.At the animal level,exosomes could down regulate the expression of oxidative stress-related markers and promote liver tissue repair.