人尿源干细胞分泌的细胞外囊泡促进后肢缺血小鼠模型中的缺血修复。
Extracellular Vesicles Secreted by Human Urine-Derived Stem Cells Promote Ischemia Repair in a Mouse Model of Hind-Limb Ischemia.
作者信息
Zhu Qingwei, Li Qing, Niu Xin, Zhang Guowei, Ling Xiaozheng, Zhang Jieyuan, Wang Yang, Deng Zhifeng
机构信息
Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
Institute of Microsurgery on Extremities, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
出版信息
Cell Physiol Biochem. 2018;47(3):1181-1192. doi: 10.1159/000490214. Epub 2018 Jul 24.
BACKGROUND/AIMS: Our previous studies have shown that human urine-derived stem cells (USCs) have great potential as a cell source for cytotherapy and tissue engineering and that extracellular vesicles (EVs) secreted by USCs (USCs-EVs) can prevent diabetes-induced kidney injury in an animal model. The present study was designed to evaluate the effects of USCs-EVs on ischemia repair.
METHODS
USCs-EVs were isolated and purified by a battery of centrifugation and filtration steps. The USCs-EVs were then characterized by transmission electron microscopy, western blot and tunable resistive pulse sensing techniques. After intramuscularly transplanting USCs-EVs into an ischemic mouse hind-limb, we observed the therapeutic effects of USCs-EVs on perfusion by laser doppler perfusion imaging, angiogenesis and muscle regeneration by histology and immunohistochemistry techniques over 21 days. We subsequently tested whether USCs-EVs can induce the proliferation of a human microvascular endothelial cell line HMEC-1 and a mouse myoblast cell line C2C12 by cell counting kit 8 assay in vitro. Meanwhile, the potential growth factors in the USCs-EVs and supernatants of the USCs cultures were detected by enzyme-linked immunosorbent assay.
RESULTS
The USCs-EVs were spherical vesicles with a diameter of 30-150 nm and expressed exosomal markers, such as CD9, CD63 and Tsg101. Ischemic limb perfusion and function were markedly increased in the hind-limb ischemia (HLI) model after USCs-EVs administration. Moreover, angiogenesis and muscle regeneration levels were significantly higher in the USCs-EVs treatment group than in the PBS group. The in vitro experiments showed that USCs-EVs facilitated HMEC-1 and C2C12 cell proliferation in a dose-dependent manner.
CONCLUSIONS
These results revealed for the first time that USCs-EVs efficiently attenuate severe hind-limb ischemic injury and represent a novel therapy for HLI.
背景/目的：我们之前的研究表明，人尿液来源的干细胞（USCs）作为细胞治疗和组织工程的细胞来源具有巨大潜力，并且USCs分泌的细胞外囊泡（USCs-EVs）可在动物模型中预防糖尿病诱导的肾损伤。本研究旨在评估USCs-EVs对缺血修复的影响。
方法
通过一系列离心和过滤步骤分离并纯化USCs-EVs。然后用透射电子显微镜、蛋白质印迹和可调电阻脉冲传感技术对USCs-EVs进行表征。将USCs-EVs肌肉内移植到缺血小鼠后肢后，我们通过激光多普勒灌注成像观察USCs-EVs对灌注的治疗效果，通过组织学和免疫组织化学技术在21天内观察血管生成和肌肉再生情况。随后，我们通过体外细胞计数试剂盒8检测法测试USCs-EVs是否能诱导人微血管内皮细胞系HMEC-1和小鼠成肌细胞系C2C12的增殖。同时，通过酶联免疫吸附测定法检测USCs-EVs和USCs培养上清液中的潜在生长因子。
结果
USCs-EVs为直径30-150nm的球形囊泡，表达外泌体标志物，如CD9、CD63和Tsg101。给予USCs-EVs后，后肢缺血（HLI）模型中缺血肢体的灌注和功能显著增加。此外，USCs-EVs治疗组的血管生成和肌肉再生水平明显高于PBS组。体外实验表明，USCs-EVs以剂量依赖的方式促进HMEC-1和C2C12细胞增殖。
结论
这些结果首次揭示，USCs-EVs能有效减轻严重的后肢缺血损伤，代表了一种治疗HLI的新疗法。
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