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中华细胞与干细胞杂志(电子版)

中华细胞与干细胞杂志(电子版) ›› 2018, Vol. 08 ›› Issue (04) : 212 -217. doi: 10.3877/cma.j.issn.2095-1221.2018.04.004

所属专题： 文献；

论著

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
陈俊秋1, 陈津2,†(), 黄梁浒1, 赵红州3, 付云烽3, 林娜3, 朱凌峰3, 程远航1, 王水良2, 谭建明1   
  1. 1. 350025　福州总医院全军器官移植研究所　（福建省移植生物学重点实验室）；350025　福州，厦门大学附属东方医院器官移植研究所
    2. 350025　福州总医院全军器官移植研究所　（福建省移植生物学重点实验室）；350025　福州，福建医科大学附属福总临床医学院；350025　福州，厦门大学附属东方医院器官移植研究所
    3. 350025　福州总医院全军器官移植研究所　（福建省移植生物学重点实验室）
  • 收稿日期:2018-03-12 出版日期:2018-08-01
  • 通信作者: 陈津
  • 基金资助:
    国家自然科学基金青年项目(81601618); 福建省自然科学基金面上项目(2016J01582、2016J01580、2018J01349); 福建省科技创新联合资金重大项目(2017Y9127)

Exosomes derived from mesenchymal stem cells promote islet endothelial cell angiogenesis

Junqiu Chen1, Jin Chen2,†(), Lianghu Huang1, Hongzhou Zhao3, Yunfeng Fu3, Na Lin3, Lingfeng Zhu3, Yuanhang Cheng1, Shuiliang Wang2, Jianming Tan1   

  1. 1. Organ Transplant Institute, Fuzhou General Hospital, Fujian Key Laboratory of Transplant Biology, Fuzhou 350025, China; Organ Transplant Institute, Dongfang Hospital, Xiamen University, Fuzhou 350025, China
    2. Organ Transplant Institute, Fuzhou General Hospital, Fujian Key Laboratory of Transplant Biology, Fuzhou 350025, China; Fuzhou General Hospital, Fujian Medical University, Fuzhou 350025, China; Organ Transplant Institute, Dongfang Hospital, Xiamen University, Fuzhou 350025, China
    3. Organ Transplant Institute, Fuzhou General Hospital, Fujian Key Laboratory of Transplant Biology, Fuzhou 350025, China
  • Received:2018-03-12 Published:2018-08-01
  • Corresponding author: Jin Chen
  • About author:
    Corresponding author: Chen Jin, Email:
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引用本文：

陈俊秋, 陈津, 黄梁浒, 赵红州, 付云烽, 林娜, 朱凌峰, 程远航, 王水良, 谭建明. 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[J]. 中华细胞与干细胞杂志(电子版), 2018, 08(04): 212-217.

Junqiu Chen, Jin Chen, Lianghu Huang, Hongzhou Zhao, Yunfeng Fu, Na Lin, Lingfeng Zhu, Yuanhang Cheng, Shuiliang Wang, Jianming Tan. Exosomes derived from mesenchymal stem cells promote islet endothelial cell angiogenesis[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2018, 08(04): 212-217.

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。

关键词: 外泌体, 低氧, 胰岛内皮细胞, 血管生成
Objective

This study was aimed to investigate the pro-angiogenesis effect of mesenchymal stem cell exosomes (MSC-exosomes) on islet endothelial cell MS-1 in hypoxia.

Methods

MSCs 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.

Results

Exosomes 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.

Conclusion

MSC-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.

Key words: Exosomes, Hypoxia, MS-1, Angiogenesis
图1 电镜下观察外泌体的形态大小（磷钨酸负染，×50K）
图2 外泌体标志物Western Blot检测鉴定
表1 Image J血管形成长度分析和VEGF相对表达量
分组 血管形成长度分析（n = 3） VEGF相对表达量（n = 8）
Nor 393.30±174.20 1.000
Hyp 1467.00±230.00a 6.52±3.50a
Hyp+EXO 2386.00±137.70ab 20.26±9.97ab
F值 87.59 24.11
P值 <0.01 <0.01
图3 倒置相差显微镜下观察MSC外泌体促进胰岛内皮细胞MS-1血管形成（×100）
图4 MSC外泌体上调血管内皮生长因子基因表达
图5 MSC外泌体上调HIF1-α表达和促进mTOR发生磷酸化
1
刘英,江霞. 糖尿病细胞治疗的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2017, 7(01):59-63.
2
Shapiro AM,Lakey JR,Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen[J]. N Engl J Med, 2000, 343(4):230-238.
3
Shapiro A,Ricordi C,Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation[J]. N Engl J Med, 2006, 355(13):1318-1330.
4
Smink AM,Faas MM,De Vos P. Toward engineering a novel transplantation site for human pancreatic islets[J]. Diabetes, 2013, 62(5):1357-1364.
5
Del Toro-Arreola A,Robles-Murillo AK,Rivas-Carrillo JD. The role of endothelial cells on islet function and revascularization after islet transplantation[J]. Organogenesis, 2016, 12(1):28-32.
6
Cao ZL,Wang XY. The endocrine role between beta cells and intra- islet endothelial cells[J]. Endocr J, 2014, 61(7):647-654.
7
Carlsson PO,Palm F,Andersson A, et al. Markedly decreased Oxygen tension in transplanted rat pancreatic islets irrespective of the implantation site[J]. Diabetes, 2001, 50(3):489-495.
8
Chen J,Ye Y,Liao L, et al. Mesenchymal stem cells promote islet survival in vitro and function in vivo[J]. CellR4, 2013, 1(2):128-136.
9
Ailawadi S,Wang X,Gu H, et al. Pathologic function and therapeutic potential of exosomes in cardiovascular disease[J]. Biochim Biophys Acta, 2015, 1852(1):1-11.
10
Chen JY,Liu ZJ,Hong MM, et al. Proangiogenic compositions of microvesicles derived from human umbilical cord mesenchymal stem cells[J]. PLoS One, 2014, 9(12):e115316.
11
Chen J,Ren Q,Cai YM, et al. Mesenchymal stem cells drive paclitaxel resistance in ErbB2/ErbB3/-coexpressing breast cancer cells via paracrine of neuregulin 1[J]. Biochem Biophys Res Commun, 2018, 501(1):212-219.
12
Chen J,Ma Y,Wang Z, et al. Thrombin promotes fibronectin secretion by bone marrow mesenchymal stem cells via the protease-activated receptor mediated signalling pathways[J]. Stem Cell Res Ther, 2014, 5(2):36.
13
Yáñez-Mó M,Siljander PR,Andreu Z, et al. Biological properties of extracellular vesicles and their physiological functions[J]. J Extracell Vesicles, 2015, 4(1):27066.
14
Zhang HC,Liu XB,Huang S, et al. Microvesicles derived from human umbilical cord mesenchymal stem cells stimulated by hypoxia promote angiogenesis both in vitro and in vivo[J]. Stem Cells Dev, 2012, 21(18):3289-3297.
15
Xu R,Simpson RJ,Greening DW. A Protocol for Isolation and Proteomic Characterization of Distinct Extracellular Vesicle Subtypes by Sequential Centrifugal Ultrafiltration[J]. Methods Mol Biol, 2017, 1545:91-116.
16
Zhang B,Yin Y,Lai RC, et al. Mesenchymal stem cells secrete immunologically active exosomes[J]. Stem Cells Dev, 2014, 23(11): 1233-1244.
17
Plock JA,Schnider JT,Zhang W, et al. Adipose- and bone Marrow-Derived mesenchymal stem cells prolong graft survival in vascularized composite allotransplantation[J]. Transplantation, 2015, 99(9):1765-1773.
18
Gai C,Carpanetto A,Deregibus MC, et al. Extracellular vesicle-mediated modulation of angiogenesis[J]. Histol Histopathol, 2016, 31(4):379-391.
19
Merino-Gonzalez C,Zuniga FA,Escudero CA, et al. Mesenchymal stem Cell-Derived extracellular vesicles promote angiogenesis: potencial clinical application[J]. Front Physiol, 2016, 7:24.
20
Anderson JD,Johansson HJ,Graham CS, et al. Comprehensive proteomic analysis of mesenchymal stem cell exosomes reveals modulation of angiogenesis via nuclear Factor-KappaB signaling[J]. Stem Cells, 2016, 34(3):601-613.
21
Tetta C,Bruno S,Fonsato V, et al. The role of microvesicles in tissue repair[J]. Organogenesis, 2011, 7(2):105-115.
22
Zhang ZW,Yang JJ,Yan WY, et al. Pretreatment of cardiac stem cells with exosomes derived from mesenchymal stem cells enhances myocardial repair[J]. J Am Heart Assoc, 2016, 5(1): pii: e002856.
23
Binet F,Sapieha P. ER stress and angiogenesis[J]. Cell Metab, 2015, 22(4):560-575.
24
Rosen R,Vagaggini T,Chen YE, et al. Zeaxanthin inhibits hypoxia-induced VEGF secretion by RPE cells through decreased protein levels of hypoxia-inducible factors-1α[J]. Biomed Res Int, 2015, 2015: 687386.
25
Karali E,Bellou S,Stellas D, et al. VEGF signals through ATF6 and PERK to promote endothelial cell survival and angiogenesis in the absence of ER stress[J]. Mol Cell, 2014, 54(4):559-572.
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