简易密封盒建立人脐静脉血管内皮细胞缺氧/复氧损伤模型

A simple sealed box for establishing hypoxia/reoxygenation injury model of human umbilical vein endothelial cells

目的:探讨简易密封盒建立人脐静脉血管内皮细胞(HUVECs)缺氧/复氧(H/R)损伤模型的可行性与重复性。方法:种于6孔板的HUVECs随机分为对照组和实验组(3 h/2 h、6 h/2 h、9 h/2 h、12 h/2 h)。对照组高糖培养基(4.5 g/L D-Glucose)培养不做其他处理,实验组细胞更换低糖培养基(1.0 g/L D-Glucose)放到简易密封盒里,并向密封盒里充入94%N_(2)、5%CO_(2)、1%O_(2)混合气体,形成缺氧环境,缺氧时间段分别为3、6、9、12 h,处理结束后,换成高糖培养基,置培养箱中培养2 h。镜下观察细胞形态、数目改变;CCK-8测定细胞增殖情况;Hoechst 33258观察细胞核形态变化;DCFH-DA染色检测活性氧自由基含量变化;Western blot检测细胞缺氧诱导因子(HIF-1α)的表达水平。结果:与对照组相比,各实验组细胞数量和密度逐渐下降;CCK-8检测显示各实验组细胞与对照组细胞相比存活率和活性均下降(P<0.05);Hoechst 33258核染色显示各实验组随着缺氧时间的延长细胞核皱缩或破裂加重;DCFH-DA染色显示各实验组随H/R时间延长,氧自由基含量增加(P<0.05);Western blot结果显示各实验组随着缺氧时间的延长HIF-1α表达逐渐增高,12 h后表达开始降低(P<0.05)。结论:利用自制的简易密封盒构建的HUVECs H/R损伤模型简单易行,重复性较好。

Objective:To investigate the feasibility and reproducibility of a simple sealed box to establish hypoxia/reoxygenation injury(H/R)model of human umbilical vein endothelial cells(HUVECs).Methods:HUVECs incubated in six-well plates were randomized to control group and experimental group(3 h/2 h,6 h/2 h,9 h/2 h,12 h/2 h).Cells in the control group were cultured in conventional high-sugar medium(containing 4.5g/L D-Glucose)without additional intervention,and those in the experimental group were cultured with low-sugar medium(containing 1.0g/L D-Glucose),and maintained in a simple sealed box in which mixed gas containing 94%N_(2),5%CO_(2) and 1%O_(2) was filled to create a hypoxic environment.The hypoxia length was set for 3,6,9,and 12 h,respectively.Upon the treatment being completed,the culture was replaced with high-sugar medium,and further cultivated in an incubator for 2 h.The cells were microscopically observed for the changes of cellular structure and number.CCK-8 kit was used to examine the cell proliferation.DCFH-DA staining and Western blot were performed to detect the change of reactive oxygen species(ROS)level,and expression level of hypoxia-inducible factor alpha(HIF-1α),respectively.Results:The number and density of HUVECs were gradually reduced in the experimental group compared to control group.CCK-8 measurement showed that the survival rate and activity of the HUVECs were decreased in the experimental group(P<0.05).Hoechst 33258 nuclear staining revealed nuclear shrinkage or cell rupture worsened with prolonged hypoxia time,and DCFH-DA staining indicated increase of ROS level with added hypoxia/reoxygenation(P<0.05).Western-blot results showed that HIF-1αexpression tended to increase with extension of hypoxia,yet was gradually down-regulated at 12 h(P<0.05).Conclusion:The hypoxia/reoxygenation injury model for HUVECs with a self-made simple sealed box can be easily established,and better reproducible.