人嗅觉黏膜间充质干细胞来源的外泌体促进内皮细胞的血管生成

外泌体作为是细胞旁分泌的重要介质，在促血管形成方面有重要作用。在我们前期研究中，已经成功从嗅黏膜间充质干细胞（olfactory mucosa mesenchymal stem cells，OM-MSCs）分离、鉴定了其外泌体，然而，OM-MSCs源外泌体对血管生成的影响尚不清楚。本研究旨在探讨OM-MSCs来源外泌体对内皮细胞血管生成能力的影响。采用PKH67 荧光标记OM-MSCs源外泌体，与人脑微血管内皮细胞（human brain microvessel endothelial cells, HBMECs) 共培养，观察 OM-MSCs外泌体能否进入 HBMECs。采用CCK-8法、Transwell 迁移实验和小管实验，观察 OM-MSCs外泌体对 HBMECs增殖、迁移及管状结构形成的影响。采用基质胶塞实验及CD31免疫荧光，观察OM-MSCs外泌体在体内对血管生成的影响。上述研究均以等量 PBS 作为对照。结果提示，OM-MSCs外泌体可被HBMECs 摄取。CCK-8 法检测显示，在处理1、2、3、4、5 d各时间点，实验组细胞增殖均优于对照组（1.32±0.14 vs. 0.98±0.04, 1.36±0.14 vs.1.04±0.06, 1.75±0.18 vs.1.33±0.11, 2.16±0.11 vs.1.50±0.19, 2.71±0.11 vs. 1.81±0.20, P<0.01）。Transwell 实验结果显示，实验组跨膜迁移细胞吸光度值较对照组显著增多（1.12±0.05 vs.0.02±0.02, P<0.05）。在体外小管实验中，从节点、交叉点、网眼数、血管分支数和总长度5个方面，实验组均高于空白对照组（374.33±127.74 vs. 193.33±44.79, 104.56±33.07 vs. 54.33±11.65, 20.11±11.20 vs. 7.56±3.64, 81.67±19.07 vs. 57.00±13.02, 11466.22±2781.03 vs. 8544.00±1848.61, P<0.05）；在体内实验中，实验组成血管及CD31阳性率（%）亦显著高于对照组（85.00±5.57 vs.8.00±2.08, P<0.05）。本研究表明：OM-MSCs外泌体可促进 HBMECs 增殖、迁移及管样结构形成，提示OM-MSCs外泌体可促进血管新生。

Exosomes from Human Olfactory Mucosa Mesenchymal Stem Cells Enhance the Angiogenesis of Endothelial Cells#br#

Exosome, as an important mediator of cell paracrine, plays an important role in promoting angiogenesis. In our preliminary studies, we have successfully isolated and identified exosomes from olfactory mucosa mesenchymal stem cells (OM-MSCs). However, the effect of OM-MSCs exosomes (OM-MSC-Exos) on angiogenesis is still unknown. This study was to evaluate OM-MSC-Exos angiogenesis promotion in vitro and in vivo. After co-culture of PKH67 fluorescently labeled OM-MSC-Exos with human brain microvessel endothelial cells (HBMECs), fluorescence microscopy had been used to observe whether OM-MSCs-Exos could absorbed by HBMECs. The proliferation, migration and effect of tubular structure formation of endothelial cells were detected by cell counting kit 8 (CCK-8), transwell and tube formation assays. Effect of OM-MSCs derived exosomes on angiogenesis in vivo was detected by the Matrigel plug assay and CD31 immunofluorescence. The same amount of PBS was used as control in the above experiments. The results showed that OM-MSCs Exos could be absorbed by HBMECs. The CCK-8 analysis showed that experimental group could promote HBMECs proliferation in processing 1, 2, 3, 4, 5 d each time point, when compared with the control group (1.32±0.14 vs. 0.98±0.04, 1.36±0.14 vs.1.04±0.06, 1.75±0.18 vs.1.33±0.11, 2.16±0.11 vs.1.50±0.19, 2.71±0.11 vs. 1.81±0.20, P<0.05). The result of Transwell assay showed that the absorbance value of trans membrane migration cells in the experimental group were significantly higher than that in the control group (1.12±0.05 vs.0.02±0.02, P<0.05). In vitro, Matrigel tube-forming experiment showed that the number of the nodes, junctions, meshes, branches and total length in the experimental group was significantly higher than that of the control group (374.33±127.74 vs. 193.33±44.79, 104.56±33.07 vs. 54.33±11.65, 20.11±11.20 vs. 7.56±3.64, 81.67±19.07 vs. 57.00±13.02, 11466.22±2781.03 vs. 8544.00±1848.61, P <0.05). In vivo, the percentage of CD31+ cells in the experimental group was significantly higher than that of the control group (85.00±5.57 vs.8.00±2.08, P <0.05). The above results indicated that the exosomes from OM-MSCs can induce the angiogenesis performance of endothelial cells.