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

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。

Inhibition of human umbilical cord mesenchymal stem cells derived exosomes on the capillarization of liver sinusoidal endothelial cells#br#

［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. ConclusionHuman umbilical cord MSC-Ex could inhibit the capillarization of LSEC in vivo and in vitro.