人脐带间质干细胞来源的外泌体抑制肝窦内皮细胞毛细血管样改变

摘要
图/表
参考文献(0)
相关文章 (15)

全文: PDF (31091 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS) 背景资料

文章导读

摘要目的: 探讨人脐带间质干细胞来源的外泌体(mesenchymal stem cell derived exosomes, MSCEx)对肝窦内皮细胞(liver sinusoidal endothelial cell, LSEC)毛细血管样改变的影响。方法: 从人脐带组织中分离培养间质干细胞，采用超速离心法提取培养上清液中的MSC-Ex。采用透射电镜、纳米颗粒跟踪分析仪以及蛋白质免疫印迹对MSC-Ex进行形态和表征的鉴定。用TNF-α诱导建立LSEC血管化模型，并加入DIO荧光染料标记的MSC-Ex共培养，观察LSEC对MSC-Ex的摄取能力。采用qRT-PCR检测LSEC中促血管生成素-2(angiopoietin-2,Ang-2)、CD34的mRNA相对表达，采用蛋白质免疫印迹检测LSEC中Ang-2、CD34蛋白表达，采用小管形成实验检测LSEC的管腔形成能力。构建蛋氨酸、胆碱缺乏饮食(methionine and choline deficiency diet，MCD)诱导的ICR小鼠肝纤维化模型，然后尾静脉分别注射MSCEx(10 mg/kg)和100 μL PBS，每3 d注射1次，共注射5次。采用天狼猩红染色及免疫组织化学染色分别观察肝组织胶原沉积及检测Ang-2蛋白表达。结果: 蛋白质免疫印迹显示MSC-Ex高表达外泌体特征性标志物Alix、TSG101、CD63、CD9，透射电镜显示MSC-Ex为直径约100 nm的盘状囊泡，纳米颗粒跟踪分析仪显示其颗粒浓度约为3.4×1011/mL。MSCEx可被LSEC摄取，并显著抑制TNF-α诱导的LSEC的管腔形成及Ang-2、血管内皮细胞标志CD34表达。天狼猩红染色和免疫组织化学染色显示，MSCEx可限制纤维化肝组织的胶原沉积和Ang-2蛋白表达。结论:人脐带MSC-Ex可在体内外抑制LSEC毛细血管样改变。

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章

关键词 ：人脐带间质干细胞, 外泌体, 肝窦内皮细胞, 毛细血管样改变, 血管生成素-2

Abstract：［Abstract］Objective： To investigate the effect of human umbilical cord mesenchymal stem cell derived exosomes(MSC-Ex) on the capillarization of liver sinusoidal endothelial cells（LSECs). Methods： Mesenchymal stem cells were isolated from human umbilical cord tissue and cultured. Subsequently, exosomes were extracted from culture supernatant by ultracentrifugation. The morphology and characterization of exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis and Western blotting. LSECs were vascularized by TNF-α in vitro, and the uptake of MSC-Ex by LSEC was observed by co-culture with red fluorescent dye DIO labeled MSC-Ex. QRT-PCR and Western blotting were used to detect the mRNA and protein expression of angiopoietin-2 （Ang-2) and CD34 in LSECs, respectively, and the tube forming ability of LSEC was detected by tubule formation test. The hepatic fibrosis model of ICR mice induced by methionine and choline deficiency diet (MCD) was established, MSC-Ex （10 mg/kg) and 100 μL PBS was injected via tail vein, once every 3 days, for a total of 5 injections. Collagen deposition and Ang-2 protein expression in liver tissue were observed by Sirius red staining and detected by immunohistochemistry, respectively. Results： Western blotting results showed that MSC-Ex expressed characteristic markers Alix, TSG101, CD63 and CD9. Transmission electron microscopy indicated that MSC-Ex was a diskshape vesicle with diameters of about 100 nm. Nano-particle analyzer showed that the particles number of exosome was about 3.4×1011/mL. MSCEx could be ingested by LSECs, and could significantly inhibited Ang2 and CD34 expression and tube formation of LSECs induced by TNF-α. Sirius red staining and immunohistochemistry indicated that MSC-Ex could inhibit collagen deposition and the protein expression of Ang-2 in fibrotic liver tissue. Conclusion：Human umbilical cord MSC-Ex could inhibit the capillarization of LSEC in vivo and in vitro.

Key words：

收稿日期: 2021-12-29

基金资助:［基金项目］江苏省社会发展重点研发计划项目(BE2020775)；江苏省研究生科研创新计划(KYCX20_3091)；镇江市社会发展重点研发计划项目(SH2020002)

通讯作者: 谭友文(通讯作者)，主任医师，硕士生导师，E-mail：tyw915@sina.com

作者简介: 王晨(1996—)，女，硕士研究生

引用本文:

王晨，王岩金，杨馥吉，等.. 人脐带间质干细胞来源的外泌体抑制肝窦内皮细胞毛细血管样改变[J]. 江苏大学学报:医学版, 2022, 32(05): 376-384. WANG Chen1,2, WANG Yanjin2, YANG Fuji2, YAN Yongmin2, TAN Youwen1. Inhibition of human umbilical cord mesenchymal stem cells derived exosomes on the capillarization of liver sinusoidal endothelial cells#br#. Journal of Jiangsu University(Medicine Edition), 2022, 32(05): 376-384.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2022/V32/I05/376

［1］Fouad Y, Esmat G, Elwakil R, et al. The egyptian clinical practice guidelines for the diagnosis and management of metabolic associated fatty liver disease［J］. Saudi J Gastroenterol, 2022, 28(1): 3-20.
［2］Wisse E, Jacobs F, Topal B,et al. The size of endothelial fenestrae in human liver sinusoids: implications for hepatocytedirected gene transfer［J］. Gene Ther, 2008, 15(17): 1193-1199.
［3］Bale SS, Geerts S, Jindal R, et al. Isolation and coculture of rat parenchymal and nonparenchymal liver cells to evaluate cellular interactions and response［J］. Sci Rep, 2016, 6: 25329.
［4］Valadi H, Ekstrom K, Bossios A, et al. Exosomemediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells［J］. Nat Cell Biol, 2007, 9(6): 654-659.
［5］Montecalvo A, Larregina AT, Shufesky WJ, et al. Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes［J］. Blood, 2012, 119(3): 756-766.
［6］施晋升，王茂松，李本忠，等. 食管癌细胞来源的外泌体促进人脐静脉血管内皮细胞血管生成［J］. 江苏大学学报(医学版)， 2021， 31(5)： 412-416， 420.
［7］倪修凡，张尤历，徐岷. 外泌体源性长链非编码RNA在消化系统肿瘤中的研究进展［J］. 江苏大学学报(医学版)， 2020， 30(4)： 293-297， 301.
［8］Puglisi MA, Tesori V, Lattanzi W, et al. Therapeutic implications of mesenchymal stemcells in liver injury［J］. J Biomed Biotechnol, 2011, 2011: 860578.
［9］Bercier P, Grenier D. TNFα disrupts the integrity of the porcine respiratory epithelial barrier［J］. Res Vet Sci, 2019, 124: 13-17.
［10］BartonPai A, Feleder C, Johnson A. Tumor necrosis factorα induces increased lung vascular permeability: a role for GSK3α/β［J］. Eur J Pharmacol, 2011, 657(1-3): 159-166.
［11］Ghoshal AK, Farber E. Choline deficiency, lipotrope deficiency and the development of liver disease including liver cancer: a new perspective［J］. Lab Invest, 1993, 68(3): 255-260.
［12］Thomas H. LSEC stretch promotes fibrosis during hepatic vascular congestion［J］. Nat Rev Gastroenterol Hepatol, 2019, 16(5): 262-263.
［13］蔡梦洁, 钱嘉莉, 陈逸菲, 等. HucMSCex通过Let7b调控TGFβR1抑制肝星状细胞胶原合成［J］. 江苏大学学报(医学版), 2018, 28(6): 478-482.
［14］Jiang W, Tan Y, Cai M, et al. Human umbilical cord MSCderived exosomes suppress the development of CCl4induced liver injury through antioxidant effect［J］. Stem Cells Int, 2018, 2018: 6079642.
［15］Hyun J, Wang S, Kim J, et al. MicroRNA125bmediated Hedgehog signaling influences liver regeneration by chorionic platederived mesenchymal stem cells［J］. Sci Rep, 2015, 5: 14135.
［16］Yang Y, Sangwung P, Kondo R, et al. Alcoholinduced Hsp90 acetylation is a novel driver of liver sinusoidal endothelial dysfunction and alcoholrelated liver disease［J］. J Hepatol, 2021, 75(2): 377-386.
［17］RodriguezVita J, MoralesRuiz M. Down the liver sinusoidal endothelial cell（LSEC) hole. Is there a role for lipid rafts in LSEC fenestration?［J］. Hepatology, 2013, 57(3): 1272-1274.
［18］Szafranska K, Kruse LD, Holte CF, et al. The whole story about fenestrations in LSEC［J］. Front Physiol, 2021, 12: 735573.
［19］Chen JX, Huang XY, Wang P, et al. Effects and mechanism of arachidonic acid against TNFα induced apoptosis of endothelial cells［J］. Clin Hemorheol Microcirc, 2021, 77(3): 259-265.
［20］Suzuki Y, Deguchi M, Furuya A, et al.Attenuates the induction of vascular cell adhesion protein 1 by TNFα in human umbilical vein endothelial cells［J］. Pharmacology, 2021, 106(3-4): 218-224.
［21］Tasev D, Konijnenberg LSF, AmadoAzevedo J, et al. CD34 expression modulates tubeforming capacity and barrier properties of peripheral bloodderived endothelial colonyforming cells［J］. Angiogenesis, 2016, 19(3): 325-338.
［22］Chen Z, Xie Y, Chen W, et al. microRNA67855ploaded human umbilical cord mesenchymal stem cellsderived exosomes suppress angiogenesis and metastasis in gastric cancer via INHBA［J］. Life Sci, 2021, 284: 119222.
［23］Liu X, Hu J, Li Y, et al. Mesenchymal stem cells expressing interleukin18 inhibit breast cancer in a mouse model［J］. Oncol Lett, 2018, 15(5): 6265-6274.
［24］Li T, Yan Y, Wang B, et al. Exosomes derived from human umbilical cord mesenchymal stem cells alleviate liver fibrosis［J］. Stem Cells Dev, 2013, 22(6): 845-854.
［25］MelgarLesmes P, Tugues S, Ros J, et al. Vascular endothelial growth factor and angiopoietin2 play a major role in the pathogenesis of vascular leakage in cirrhotic rats［J］. Gut, 2009, 58(2): 285-292.
［26］Kapiainen E, Kihlstrm MK, Pietil R, et al. The aminoterminal oligomerization domain of angiopoietin2 affects vascular remodeling, mammary gland tumor growth, and lung metastasis in mice［J］. Cancer Res, 2021, 81(1): 129-143.
［27］Zhang ZL, Zhang JF, Yuan YF, et al. Suppression of angiogenesis and tumor growth in vitro and in vivo using an antiangiopoietin2 singlechain antibody［J］. Exp Ther Med, 2014, 7(3): 543-552.
［28］Sun B, Dong C, Lei H, et al. Propranolol inhibits proliferation and induces apoptosis of hemangiomaderived endothelial cells via Akt pathway by downregulating Ang2 expression［J］. Chem Biol Interact, 2020, 316: 108925.
［29］Lefere S, Van de Velde F, Hoorens A, et al.Angiopoietin2 promotes pathological angiogenesis and is a therapeutic target in murine nonalcoholic fatty liver disease［J］. Hepatology, 2019, 69(3): 1087-1104.
［30］Deleve LD, Wang X, Guo Y. Sinusoidal endothelial cells prevent rat stellate cell activation and promote reversion to quiescence［J］. Hepatology, 2008, 48(3): 920-930.