中国口腔颌面外科杂志

中国口腔颌面外科杂志 ›› 2019, Vol. 17 ›› Issue (4): 295-299.doi: 10.19438/j.cjoms.2019.04.002

• 论著 • 上一篇    下一篇

人血管内皮细胞缺氧复氧损伤细胞模型的建立

牛其芳1,2, 李德龙1, 杨杨1, 秦力铮1, 冯芝恩1, 李金忠1, 韩正学1   

  1. 1.首都医科大学附属北京口腔医院 口腔颌面-头颈肿瘤外科,北京 100050;
    2.首都医科大学附属复兴医院 口腔科,北京 100037
  • 收稿日期:2018-11-12 修回日期:2019-01-16 出版日期:2019-07-20 发布日期:2019-08-12
  • 通讯作者: 韩正学,E-mail：hanf1989@hotmail.com
  • 作者简介:牛其芳（1990-）,女,硕士,医师,现工作于首都医科大学附属复兴医院口腔科,E-mail：zqnqf@163.com
  • 基金资助:
    国家自然科学基金（81570957）; 北京口腔医院学科建设基金（18-09-21）

Establishment of human vascular endothelial hypoxia/reoxygeneration injury cell model

NIU Qi-fang1,2, LI De-long1, YANG Yang1, QIN Li-zheng1, FENG Zhi-en1, LI Jin-zhong1, HAN Zheng-xue1   

  1. 1.Department of Oromaxillofacial Head and Neck Oncology, Beijing Stomatological Hospital, Capital Medical University. Beijing 100050;
    2.Department of Stomatology, Fuxing Hospital, Capital Medical University. Beijing 100037, China
  • Received:2018-11-12 Revised:2019-01-16 Online:2019-07-20 Published:2019-08-12

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摘要： 目的 构建人血管内皮细胞缺氧复氧细胞模型,作为组织瓣缺血再灌注损伤的体外研究模型。方法 原代人脐静脉内皮细胞（HUVECs）培养扩增至第5代。依据缺氧时间随机分为4组：正常对照组（Control组）,3h/2h组、6h/2h组、9h/2h组;Control组以完全培养基正常培养,实验组分别以低糖无血清培养基于缺氧环境下培养3、6、9 h,后换用原完全培养基复氧培养2 h。显微镜下大体观察各组细胞形态、结构、数目,台盼蓝实验测定存活细胞比例,CCK-8法测定细胞活性,Annexin V-PI 染色流式细胞仪测定细胞凋亡比例。采用SPSS 19.0软件包对数据进行统计学分析。结果 与对照组相比,镜下可见实验组细胞损伤程度明显较高;随缺氧时间延长,损伤加重,细胞密度减低。台盼蓝实验及CCK-8实验结果显示,实验组存活细胞比例及细胞活性较对照组显著降低,且随缺氧时间增加而显著下降（P<0.05）。细胞凋亡实验表明,随缺氧时间增加,细胞早期凋亡、晚期凋亡比例均显著上升（P<0.05）,细胞凋亡水平加重。结论 建立了稳定可靠的人血管内皮细胞缺氧/复氧损伤细胞模型,为组织瓣缺血再灌注损伤的体外实验研究提供了基础。

关键词: 血管内皮细胞, 缺氧/复氧模型, 组织瓣, 缺血再灌注损伤

Abstract: PURPOSE: To establish a cell model of human vascular endothelial hypoxia/reoxygenation injury, as an in vitro model of tissue flap ischemia-reperfusion injury (IRI). Methods: Primary human umbilical vein endothelial cells (HUVECs) were cultured to the 5th generation, then divided into 4 groups randomly according to the time of exposing in hypoxia condition as 3h/2h group, 6h/2h group, 9h/2h group and control group. The cells of hypoxia groups were placed in low-glucose and serum-free DMEM and submitted to hypoxia condition for 3, 6 and 9 h respectively,then cultured in normoxia condition with original complete medium for 2 h as reoxygenation. After reoxygenation, morphological features, structures and numbers were observed, the rate of surviving cells was determined by Trypan blue assay, and the cell viability was measured by CCK-8 assay, the rate of apoptotic cells was detected by flow cytometry with Annexin V-PI assay. The data were analyzed with SPSS 19.0 software package. Results: Compared with the control group,the morphology of HUVECs were obviously damaged in HRI groups,with increasing time of hypoxia,the damage of cells gradually aggravated. In Trypan blue and CCK - 8 assays, the rate of surviving cells and the cell viability decreased in HRI groups, with the increase of hypoxia time, the rate of surviving cells and the cell viability decreased significantly (P<0.05). Annexin V-PI assay showed that the early and late apoptotic rate of cells in HRI groups were significantly higher compared with the control group; and with the increase of hypoxia time,the early and late apoptotic rate increased gradually(P<0.05). Conclusions: The reliable cell model of human vascular endothelial hypoxia/reoxygenation injury was established successfully with cultured HUVECs, which provides a basis for in vitro studies of tissue flap ischemia-reperfusion injury.

Key words: Vascular endothelial cells, Hypoxia/reoxygenation injury model, Tissue flap, Ischemia reperfusion injury

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