人嗅黏膜间充质干细胞来源外泌体的分离鉴定及生物学特性研究

外泌体是指释放到细胞外微环境中的直径约50～130 nm的纳米级的膜性囊泡。嗅黏膜间充质干细胞（olfactory mucosa mesenchymal stem cells，OM-MSCs）作为一类新发现的间充质干细胞，在许多疾病中均具有治疗作用，且其内在机制与其旁分泌的外泌体密切相关，但OM-MSCs外泌体的分离、鉴定及生物学特性的研究尚未见报道。本研究采用超速离心法提取OM-MSCs培养液中的外泌体，应用流式细胞术及免疫荧光进行细胞鉴定后，分别用透射电子显微镜、纳米粒径分析及Western印迹对外泌体形态、颗粒大小和表面的特异性分子标志进行分析鉴定。采用CCK8增殖实验，Western印迹和划痕实验，分析其对人脑微血管内皮细胞增殖和迁移的影响。电镜、Western 印迹和纳米粒径分析的结果显示：OM-MSCs来源外泌体形态多为圆形，直径约为40～150 nm；表达外泌体标记物CD63，CD81；CCK-8法检测显示：不同浓度的OM-MSCs源外泌体可提高人脑微血管内皮细胞的增殖活性，且其增殖促进作用具有浓度依赖性（P<0.05）。Western 印迹检测结果显示：相比空白对照组，OM-MSCs源外泌体可显著提高内皮细胞的增殖细胞核抗原蛋白质水平表达（P<0.01），细胞划痕实验结果显示，OM-MSCs源外泌体可增强内皮细胞的迁移能力，且高于对照组（P<0.01）。本研究表明：通过超速离心法可以分离纯化获得OM-MSCs源外泌体，且该外泌体具有促进人脑微血管内皮细胞迁移和增殖的作用。

Isolation,Identification and Biological Characterization of Exosomes Derived from Human Olfactory Mucosa Mesenchymal Stem Cells

Exosomes released into the extracellular microenvironment are nanoscale membrane vesicles with a diameter of 30-150 nm．As a kind of newly discovered mesenchymal stem cells, olfactory mucosa mesenchymal stem cells (OM-MSCs) are potential therapeutic targets for various diseases, and the mechanism of action is postulated to be mediated by secretion of paracrine factors-exosomes. However, the studies for isolation, identification and biological characterization of OM-MSC exosomes remain unreported. In this study, ultracentrifugation was used to isolate exosomes from conditioned medium of OM-MSCs. After the OM-MSCs were isolated and cultured, we used flow cytometry and immunofluorescence for identification. Transmission electron microscopy, nanoparticle size analysis and Western blotting were used to analyze and identify the shape, particle size and specific surface molecular markers of exosomes, respectively. CCK8 proliferation tests, Western blotting and scratch tests were used to analyze the effects of exosomes on the proliferation and migration of human brain microvessel endothelial cells (HBMECs). Our results showed that the OM-MSC exosome morphology was mostly round with a diameter of 40-150 nm, and they expressed exosome markers CD63 and CD81. Moreover, OM-MSC exosomes could significantly promote HBMEC proliferation and migration. This study demonstrates that exosomes can be successfully isolated and purified from the supernatant of OM-MSCs by ultracentrifugation and these exosomes can significantly promote the proliferation and migration of HBMECs.