南方医科大学学报

南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (8): 1905-1914.doi: 10.12122/j.issn.1673-4254.2026.08.18

• • 上一篇    

常氧/缺氧人脐静脉内皮细胞来源的外泌体对心肌损伤和成纤维活化的抑制作用及其机制

熊小栓1(), 杨涛1, 陈俊羽2, 杨晓晴2, 陈史钰2, 阮焕钧2, 王小庆2, 张炜宁1, 程芳洲1, 柯晓2,3()   

  1. 1.深圳市盐田区人民医院心内科，广东 深圳 518081
    2.中国医学科学院阜外医院深圳医院心内科，广东 深圳 518057
    3.深圳心血管疾病重点实验室//中国医学科学院阜外医院深圳医院，广东 深圳 518057
  • 收稿日期:2026-01-08 出版日期:2026-08-20 发布日期:2026-08-01
  • 通讯作者: 柯晓 E-mail:155256040@qq.com;xiaokehospital@126.com
  • 作者简介:熊小栓，硕士，副主任医师，E-mail: 155256040@qq.com
  • 基金资助:
    广东省自然科学基金区域联合基金-地区培育项目(2023A1515140174);广东省基础与应用基础研究基金(2026A1515010655);深圳市科技计划项目(JCYJ20230807150802005);深圳市科技计划项目(JCYJ20230807150803007);深圳市盐田区卫生科技计划项目(YTWS20220209)

Effects of exosomes derived from normoxic or hypoxic human umbilical vein endothelial cells on hypoxia-induced cardiomyocyte injury and cardiac fibroblast activation in vitro

Xiaoshuan XIONG1(), Tao YANG1, Junyu CHEN2, Xiaoqing YANG2, Shiyu CHEN2, Huanjun RUAN2, Xiaoqing WANG2, Weining ZHANG1, Fangzhou CHENG1, Xiao KE2,3()   

  1. 1.Department of Cardiovascular Medicine, Shenzhen Yantian District People's Hospital, Shenzhen 518081, China
    2.Department of Cardiology, Fuwai Shenzhen Hospital, Chinese Academy of Medical Sciences, Shenzhen 518057, China
    3.Shenzhen Key Laboratory of Cardiovascular Disease, Fuwai Shenzhen Hospital, Chinese Academy of Medical Sciences, Shenzhen 518057, China
  • Received:2026-01-08 Online:2026-08-20 Published:2026-08-01
  • Contact: Xiao KE E-mail:155256040@qq.com;xiaokehospital@126.com

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摘要：

目的 探究人脐静脉内皮细胞（HUVECs）在常氧和缺氧环境下产生的外泌体（N-exo和H-exo）对缺氧诱导的人心肌细胞AC16细胞损伤和大鼠心脏成纤维细胞 （RCFs）活化的作用和机制。 方法 缺氧培养24 h诱导AC16细胞损伤和RCFs活化。差速离心法和蔗糖密度梯度离心法提取HUVECs来源的外泌体，利用透射电子显微镜、纳米颗粒跟踪分析技术、考马斯亮蓝染色和Western blotting检测外泌体标志蛋白对外泌体进行鉴定。分别设置对照组：AC16和RCFs正常培养；Hypoxia组：1% O2培养24 h；N-exo/H-exo组：N-exo/H-exo干预24 h；Hypoxia+N-exo/H-exo组：缺氧培养同时加入外泌体干预；Western blotting法检测蛋白表达水平；RT-qPCR检测mRNA表达水平；荧光探针DCFH-DA检测AC16细胞内活性氧水平；TUNEL法检测AC16细胞凋亡；EDU法检测RCFs的增殖能力。 结果 N-exo和H-exo电镜下为典型的茶托型双层囊泡状；N-exo和H-exo的浓度分别为1.5×109 mL-1和7.2×108 mL-1；考马斯亮蓝染色显示HUVEC-exo具有丰富的蛋白谱；HUVECs-exo中可检测出外泌体标志性蛋白HSP70条带；与对照组相比，Hypoxia组AC16细胞中CK-MB的mRNA表达水平上升，缝隙连接蛋白43（Cx43）和谷胱甘肽过氧化酶4（GPX4）的mRNA表达水平下降，ALP、IL-1β和cTnT的蛋白表达水平上升，细胞中活性氧含量升高，细胞凋亡增加（P<0.05）；与Hypoxia组相比，107 mL-1 N-exo组CK-MB水平降低，Cx43和GPX4水平上升，ALP、IL-1β和cTnT水平下降，细胞中活性氧含量降低，细胞凋亡减少（P<0.05）；而H-exo的作用与N-exo相反（P<0.05）。与对照组相比，Hypoxia组RCFs细胞中I型胶原蛋白（Collagen I）、III型胶原蛋白（Collagen III）和α-平滑肌肌动蛋白（α-SMA）的表达水平上升，增殖能力提高（P<0.05）；与Hypoxia组相比，107 mL-1的N-exo组上述指标均降低（P<0.05）；而H-exo的作用与N-exo相反（P<0.05）。与对照组相比，Hypoxia组的AC16和RCFs中的缺氧诱导因子1亚基α（HIF-1α）和血管内皮生长因子（VEGF）水平上升，蛋白激酶B（AKT）磷酸化水平上升（P<0.05）；而与Hypoxia组相比，N-exo组的上述指标下降（P<0.05），H-exo组的上述指标上升（P<0.05）；HIF-1α抑制剂Px-478对缺氧诱导的AC16细胞损伤和RCFs细胞活化有抑制作用（P<0.05）。 结论 常氧环境下HUVECs来源的外泌对缺氧诱导的AC16细胞损伤和RCFs活化有抑制作用，缺氧环境下HUVECs来源的外泌体则加重缺氧诱导的AC16细胞损伤和RCFs活化，该作用机制可能涉及HIF-1α信号通路。

关键词: 人脐静脉内皮细胞, 心脏成纤维细胞, 心肌细胞, 缺氧诱导因子

Abstract:

Objective To investigate the effects of exosomes derived from human umbilical venous endothelial cells (HUVECs) cultured in normoxic and hypoxic conditions (N-exo and H-exo, respectively) on hypoxia-induced injury of human cardiomyocyte AC16 cells and activation of rat cardiac fibroblasts (RCFs) in vitro. Methods N-exo and H-exo were extracted from HUVECs by differential centrifugation and sucrose density gradient centrifugation and characterized. AC16 cells and RCFs in normoxic culture or hypoxic culture (1% O₂ for 24 h) were treated with N-exo or H-exo, and the changes in protein and mRNA levels were examined with Western blotting and RT-qPCR. Intracellular ROS levels and apoptosis in AC16 cells were assessed by DCFH-DA staining and TUNEL staining, and the changes in proliferation of RCFs were evaluated with EdU assay. Results N-exo and H-exo with densities of 1.5×10⁹ and 7.2×10⁸ mL-1 were obtained, respectively, which showed abundant protein components and expressed the exosomal marker HSP70. In AC16 cells, hypoxic exposure significantly upregulated CK-MB mRNA and downregulated Cx43 and GPX4 mRNA levels, causing also enhanced expressions of ALP, IL-1β and cTnT proteins and increased intracellular ROS content and cell apoptosis. These changes were effectively alleviated by treatment with 10⁷ mL-1 N-exo but worsened after treatment with H-exo. In RCFs, hypoxia significantly increased the expressions of collagen I, collagen III and α‑SMA and cell proliferation, which were suppressed by N-exo treatment but further enhanced by H-exo treatment. Hypoxia exposure increased HIF-1α and VEGF expression and phosphorylation level of AKT in both AC16 cells and RCFs; N-exo obviously inhibited while H-exo further enhanced these changes. Px-478, a specific HIF-1α inhibitor, significantly inhibited hypoxia-induced AC16 cell injury and RCF activation. Conclusion N-exo derived from HUVECs inhibits while H-exo exacerbates hypoxia-induced AC16 cell injury and RCF activation possibly via the HIF-1α pathway.

Key words: human umbilical vein endothelial cells, cardiac fibroblasts, cardiomyocyte, hypoxia-inducible factor