INTRODUCTION This study assessed whether mesenchymal stem cell (MSC)-derived extracellular vesicles influenced ageing and pluripotency markers in cell cultures where they are added. METHODS MSC-derived extracellular vesicles from old and young rat bone marrows were isolated by ultracentrifugation and were characterised by western blotting, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). They were added to young and old MSC cultures. Real-time quantitative reverse transcription polymerase chain reactions and western blot analysis were performed to check the markers of ageing (vinculin and lamin A), pluripotency markers (Nanog and Oct4) and components of the mTOR signalling pathway (Rictor, Raptor, AKT and mTOR) in these cell populations. Subsequently, microRNA (miR)-188-3p expression was transiently inhibited in young MSCs to demonstrate the influence of mTOR2 on MSC ageing. RESULTS Incubation with young MSC-derived extracellular vesicles decreased the levels of ageing markers and components of the mTOR pathway and increased the pluripotency markers from old MSC populations. By contrast, incubation of young MSCs with old MSC-derived extracellular vesicles generated the reverse effects. Inhibition of miR-188-3p expression in young MSCs produced extracellular vesicles that when incubated with old MSCs produced an increase in the levels of Rictor, as well as a decrease of phosphor-AKT, as indicated by a significant decrease in beta-galactosidase staining. CONCLUSIONS MSC-derived extracellular vesicles affected the behaviour of MSC cultures, based on their composition, which could be modified in vitro. These experiments represented the basis for the development of new therapies against ageing-associated diseases using MSC-derived extracellular vesicles.

中文翻译：

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骨髓间充质干细胞来源的细胞外囊泡的影响及其在衰老中的作用。

引言这项研究评估了间充质干细胞（MSC）衍生的细胞外囊泡是否会影响添加了它们的细胞培养物中的衰老和多能性标志物。方法采用超速离心法分离老龄和幼年大鼠骨髓中的MSC细胞外囊泡，并用western blotting，纳米粒子跟踪分析（NTA）和透射电镜（TEM）进行表征。它们被添加到年轻和古老的MSC文化中。进行实时定量逆转录聚合酶链反应和蛋白质印迹分析，以检查衰老标记（vinculin和层粘连蛋白A），多能标记（Nanog和Oct4）和mTOR信号传导途径的组成部分（Rictor，Raptor，AKT和mTOR） ）在这些细胞群中。后来，microRNA（miR）-188-3p表达在年轻的MSC中被瞬时抑制，以证明mTOR2对MSC衰老的影响。结果与年轻的MSC来源的细胞外囊泡一起孵育可降低衰老标志物和mTOR通路成分的水平，并增加了来自老MSC群体的多能性标志物。相比之下，将年轻的MSC与旧的MSC衍生的细胞外囊泡孵育会产生相反的作用。在年轻的MSC中抑制miR-188-3p的表达会产生细胞外囊泡，当与旧的MSC孵育时，Rictor的水平会增加，磷光蛋白的AKT也会减少，这由β-半乳糖苷酶染色的显着降低表明。结论MSC衍生的细胞外囊泡基于其成分影响了MSC培养物的行为，可以在体外进行修改。这些实验代表了使用源自MSC的细胞外囊泡开发新的抗衰老相关疾病疗法的基础。