人血管内皮细胞缺氧复氧损伤细胞模型的建立

Establishment of human vascular endothelial hypoxia/reoxygeneration injury cell model

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。

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 5 th generation,then divided into 4 groups randomly according to the time of exposing in hypoxia condition as 3 h/2 h group,6 h/2 h group,9 h/2 h 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.