间充质细胞外泌体促进小鼠胰岛内皮细胞血管生成的研究

目的探讨间充质细胞（MSC）外泌体对低氧条件下胰岛内皮细胞（MS-1）血管生成的影响。 方法MSC无血清低氧条件培养48?h，超滤离心法富集条件培养基中的外泌体，采用电镜和Western Blot的方法进行鉴定；通过血管形成试验比较分析不同条件下：常氧培养组（NOR组，21﹪O₂、5﹪CO₂）、低浓度氧培养组（HYP组，2﹪O₂、5﹪CO₂）、外泌体+低浓度氧共培养组（HYP+EXO组，2﹪O₂、5﹪CO₂），MS-1细胞的血管形成能力；image J软件分析血管形成长度；PCR、Q-PCR检测血管内皮生长因子（VEGF） RNA水平的表达，Western Blot检测VEGF、HIF1α蛋白水平表达以及mTOR信号通路激活情况。采用单因素方差分析和SNK-q检验统计学分析。 结果超滤离心法富集的MSC条件培养基中的外泌体，大小为30 ~ 100 nm，表达CD9，CD63，CD81等外泌体表面标志物；血管形成试验结果显示，低氧促进MS-1血管生成，HYP+EXO组形成明显的血管网状结构；HYP+EXO组血管形成相对长度（2386.0±137.7）像素与NOR组（393.3±174.2）像素和HYP组（1467.0±230.0）像素相比增强，差异有统计学意义（t = 12.30，P?= 0.0065；t = 15.74，P = 0.0040）；PCR结果显示，HYP+EXO组VEGF相对表达量（20.26±9.972）较常氧对照组（1.000）和低氧组（6.521±3.501）均增强，差异有统计学意义（t = 5.462，P = 0.0009；t = 4.238，P = 0.0038）；同时，Western Blot结果显示VEGF蛋白水平表达升高，HIF1-α表达上调，mTOR发生磷酸化。 结论MSC外泌体可促进低氧条件下的小鼠胰岛内皮细胞血管生成。MSC外泌体可能通过上调HIF1-α，调节VEGF表达，激活mTOR信号通路，促进胰岛内皮细胞血管生成。

Exosomes derived from mesenchymal stem cells promote islet endothelial cell angiogenesis

ObjectiveThis study was aimed to investigate the pro-angiogenesis effect of mesenchymal stem cell exosomes (MSC-exosomes) on islet endothelial cell MS-1 in hypoxia. MethodsMSCs were cultured with serum free medium in hypoxia condition for 48 h. The MSC- exosomes in conditioned medium were enriched by centrifugal ultrafiltration, and then characterized by electron microscopy and WB analyses. The tube formation assay was performed to assesse pro-angiogenic activity of MSC-exosomes on MS-1 in normoxia (21﹪ O₂, NOR), hypoxia (2﹪ O₂, HYP) or hypoxia in the presence of exosomes (HYP+EXO). The length of tubes formed was quantified by image J software. The expression of vascular endothelial growth factor (VEGF) was detected by PCR and WB. The expression of HIF1α and activation of mTOR signaling pathway were revealed by WB. The differences were assayed with one-way analysis of variance or SNK-q test. ResultsExosomes enriched from conditioned medium of MSCs were about 30?~?100 nm diameters, and expressed exosomes markers CD9, CD63, CD81 and Flotinlin1. The results of tube formation assay showed that hypoxia induced MS-1 angiogenesis, which was significantly enhanced by MSCs-exosomes. The length of tubes of the HYP+EXO group (2386.00±137.70) was increased significantly (t = 12.30, P = 0.0065; t?=15.74, P?=?0.0040) both than NOR group (393.30±174.20)pixels and HYP group (1467.00±230.00)pixels. PCR results showed that the relative expression of VEGF in HYP+EXO group (20.26±9.972) was significantly higher (t = 5.462, P = 0.0009; t = 4.238, P = 0.0038) than that in NOR-group (1.000) and HYP group (6.521±3.501). Meanwhile, Western blot results showed that HIF1-α and VEGF expression of MS-1 was up regulated and the mTOR signal pathway was activated after treated with MSC-exosomes. ConclusionMSC-exosomes could promote angiogenesis in mouse islet endothelial cells MS-1 under hypoxic condition through up-regulation of HIF1-α and VEGF expression, and activation of mTOR signaling pathway.