缺氧H9c2来源外泌体对HUVEC增殖、迁移和成管能力的影响

Effects of hypoxia H9c2 exosome on proliferation,migration and tube formation of HUVEC

目的探讨缺氧状态下大鼠心肌细胞H9c2来源外泌体对血管新生的影响。方法采用混合气体法制备H9c2细胞缺氧模型,以正常培养细胞为对照。分别提取2组细胞分泌的外泌体,采用纳米颗粒跟踪分析检测外泌体的浓度和粒径,透射电子显微镜观察外泌体的形态和大小,Western blot验证外泌体标志蛋白。制备人脐静脉内皮细胞(HUVEC)缺氧模型,并与H9c2外泌体共同孵育,分为常氧组、缺氧组、缺氧+正常H9c2外泌体(EXO-C)组和缺氧+缺氧H9c2外泌体(EXO-M)组。通过CCK-8法、细胞划痕实验、Matrigel体外三维成形实验分别检测HUVEC增殖、迁移、成管能力。结果外泌体鉴定结果显示常氧组和缺氧组H9c2外泌体样本颗粒浓度均处于1×10^(7)×10^(12)/mL,粒径大小40~160 nm;形态特征均为球形或茶托状结构,大小均一,形态完整;外泌体标志蛋白肿瘤易感基因101(TSG101)、CD63、CD9均有表达,无阴性蛋白钙联蛋白(Calnexin)的表达。与常氧组比,缺氧组HUVEC增殖能力、迁移面积、迁移率均显著下降,参与成管的长度及分支数、节点数均减少(P<0.01);与缺氧组比,EXO-M组HUVEC细胞增殖能力下降,迁移面积减少、迁移率下降,参与成管长度及分支数进一步减少(P<0.05);与EXO-C组相比,EXO-M组增殖能力下降,细胞迁移面积减少,迁移率下降(P<0.01)。结论缺氧H9c2来源的外泌体对HUVEC增殖、迁移、成管能力有一定程度的抑制作用。

Objective To investigate the role of H9c2-derived exosomes in regulating angiogenesis in rat cardiomyocytes under hypoxia.Methods The hypoxia model of H9c2 cells was prepared by mixed gas method(the hypoxia model group),and the normal cultured cells were used as the control group.The exosomes secreted by the two groups of cells were extracted respectively.The concentration and particle size of exosomes were detected by nanoparticle tracking analysis.The morphology and size of exosomes were detected by transmission electron microscopy.Western blot assay was used to verify the exosome marker proteins.The hypoxia model of human umbilical vein endothelial cells(HUVEC)was established.HUVECs were incubated with H9c2 exosomes and divided into the normoxia group,the hypoxia group,the hypoxia+normal H9c2 exosomes(EXO-C)group and the hypoxia+hypoxia H9c2 exosomes(EXO-M)group.The proliferation,migration and tube formation of HUVECs were detected by CCK-8 method,cell scratch test and Matrigel in vitro three-dimensional forming test.Results The results of exosome identification showed that the particle concentration of H9c2 exosome samples was 1×10^(7)-1×10^(12) particles/mL and the particle size was 40-160 nm in the normoxia group and the hypoxia group.The morphological characteristics were spherical or saucer-like structure,uniform in size and complete in shape.Exosome marker proteins TSG101,CD63 and CD9 were expressed,and there was no expression of negative protein Calnexin.Compared with the normoxic group,the proliferation ability,migration area and migration rate of HUVEC were significantly decreased in the hypoxic group,and the length of tube,the number of branches and the number of nodes were decreased(P<0.01).Compared with the hypoxia group,the proliferation ability of HUVEC cells was decreased,the migration area was decreased,the migration rate was decreased and the length and number of branches involved in tube formation were further decreased in the EXO-M group(P<0.05).Compared with the EXO-C group,the proliferation ability of the EXO-M group decreased,the cell migration area decreased and the migration rate decreased(P<0.01).Conclusion Exosomes derived from hypoxic H9c2 can inhibit the proliferation,migration and tube formation of HUVEC.