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中国应用生理学杂志 ›› 2016, Vol. 32 ›› Issue (5): 390-394.doi: 10.13459/j.cnki.cjap.2016.05.002

• 研究论文 • 上一篇    下一篇

离体大鼠主动脉内皮细胞氧化应激损伤模型的建立及评价

武磊1, 周学思1, 杲修杰1, 杨苗苗1,2, 张志清1, 李景刚3, 王天辉1   

  1. 1. 军事医学科学院卫生学环境医学研究所, 天津 300050;
    2. 天津体育学院, 天津 300381;
    3. 空军后勤部疾病预防控制中心, 北京 100076
  • 收稿日期:2015-08-13 修回日期:2016-06-13 出版日期:2016-09-28 发布日期:2018-06-20
  • 通讯作者: 王天辉,Tel:022-84655322;E-mail:wydny668@163.com E-mail:wydny668@163.com
  • 基金资助:
    国家自然科学基金课题(81373108,30971421)

Establishment and evaluation of oxidative stress injury model of in vitro rat aortic endothelial cells

WU Lei1, ZHOU Xue-si1, GAO Xiu-jie1, YANG Miao-miao1,2, ZHANG Zhi-qing1, LI Jing-gang3, WANG Tian-hui1   

  1. 1. Institute of Health and Environmental Medicine, Academy of Military Medical Sciences, Tianjin 300050;
    2. Tianjin University of Sport, Tianjin 300381;
    3. Air Force Logistics Center for Disease Control and Prevention, Beijing 100076, China
  • Received:2015-08-13 Revised:2016-06-13 Online:2016-09-28 Published:2018-06-20
  • Supported by:
    国家自然科学基金课题(81373108,30971421)

摘要: 目的:建立离体大鼠主动脉内皮细胞氧化应激损伤模型,为细胞损伤及细胞凋亡的调控研究提供基础。方法:大鼠断头处死在无菌条件下开胸取主动脉,经组织块培养法后传代培养得到充足主动脉内皮细胞,接种于96孔板或爬片培养,每组设6个复孔,用于之后的各项试验检测。以不加H2O2的组作为对照组,以不同浓度的H2O2(100、200、300、400、500 μmol/L)作用于内皮细胞相同时间12 h,来筛选最佳作用浓度;依据结果以相同浓度的H2O2(100和200 μmol/L)分别作用不同时间(3、6、9、12及24 h),来筛选最佳作用时间。通过免疫荧光法鉴定、细胞存活率检测、生化指标(LDH-L、NO、MDA、SOD)检测及内皮细胞凋亡指数等变化,评价及验证模型的建立。结果:对细胞内Ⅷ型胶原抗原进行免疫荧光染色后鉴定血管内皮细胞培养成功;在12 h的相同作用时间下,随着H2O2浓度的加大,细胞存活率呈显著下降(77.63%±5.20%~40.90%±2.10%);相同浓度(100 μmol/L组和200 μmol/L组)随着作用时间的增加,细胞存活率呈显著递减(100 μmol/L组为86.83%±12.11%~44.26%±5.70%,200 μmol/L组为78.28%±11.98%~34.45%±5.87%);以H2O2浓度为100 μmol/L作用3、6、9、12及24 h,培养液中生化指标在9 h后LDH-L与MDA呈显著递增,NO与SOD呈显著递减;在H2O2浓度为100 μmol/L与作用时间12 h的条件下,流式检测结果显示内皮细胞凋亡率为16.92%±2.37%,显著高于对照组2.68%±0.47%(P<0.01); TUNEL检测内皮细胞凋亡指数为17.65%±2.36%,显著高于对照组的3.23%±0.57%(P<0.01)。结论:该方法成功建立了体外血管内皮细胞氧化应激损伤模型,探索了轻重适度的诱导细胞损伤和细胞凋亡的造模方法,可以成为开展多种血管内皮细胞损伤及凋亡调控机制研究的基础。

关键词: 大鼠, 内皮细胞, 氧化应激, 损伤, 凋亡, 模型

Abstract: Objective: To establish a model of oxidative stress injury in cultured rat aortic endothelial cells, and to provide a basis for the research of cell injury and apoptosis. Methods: The rats were decapitated to get the aorta in thoracic operation under aseptic conditions. By subculture after tissue block culture method to get sufficient aortic endothelial cells, cultured in 96-well plates or grow on cover glass for the following test. Without H2O2 group as a control group, with different doses of H2O2 (100,200,300,400,500 μmol/L) treated endothelialcells in 12 h to screen the optimal dose. Based on the results, with the same dose of H2O2 (100 or 200 μmol/L) acted on endothelial cells respectively in different time (3, 6, 9, 12 and 24 h) to screen the optimal duration. Each group was made in sextuplicate. The establishment of the model was evaluated by immunofluorescence,cell viability testing, biochemical indicators detection (lactate dehydrogenase(LDH), nitric oxide(NO), malondialdehyde(MDA), superoxidedismutase(SOD))and apoptosis index testing. Results: Endothelial cells were cultured successfully and verified by immunofluorescence staining of intracellular antigen Ⅷ collagen. With the increase of H2O2 doses at the same action time 12 h, the cell viability was significantly decreased (77.63%±5.20% to 40.90%±2.10%). The same dose(100 μmol/L group and 200 μmol/L group)with the action time increasing, the cellviability was significantly decreased (100 μmol/L group was 86.83%±12.11% to 44.26%±5.70%, 200 μmol/L group was 78.28%±11.98% to 34.45%±5.87%). At dose of H2O2 was 100 μmol/L and treated in 3,6,9,12 and 24 h, LDH-L and MDA were significantly increased after 9 h while NO and SOD were significantly decreased. In H2O2 dose of 100 μmol/L and action time 12 h, flow cytometry showed endothelial cellapoptosis rate was 16.92%±2.37%, significantly higher than the control group of 2.68%±0.47%(P<0.01); TUNEL detected endothelial cell apoptosis index was17.65%±2.36%, which was significantly higher than that in the control group of 3.23%±0.57%(P<0.01). Conclusion: The method was successfullyestablished a model of oxidative stress injury in cultured rat aortic endothelial cells, explore the moderate conditions that induced cells injury and apoptosis which could be a basis for the research.

Key words: rats, endothelial cells, oxidative stress, injury, apoptosis, model

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