| 引用本文:胡艳红1，雷洪涛2，王淑艳1，于雪1，张赛1，苏靖1，马家宝1，姜昭妍1，张凡1，万亮琴1，臧妍妍1，李芳赫1，李卫红1.缺血再灌注损伤脑微血管内皮细胞Necroptosis模型的建立[J].世界中医药,2017,(04):. |
|
| 缺血再灌注损伤脑微血管内皮细胞Necroptosis模型的建立 |
| The Establishment of Necroptosis Model on Mimic Ischemic Reperfusion Injury in Brain Microvascular Endothelial Cells |
| 投稿时间：2016-08-16 |
| DOI：10.3969/j.issn.1673-7202.2017.04.043 |
| 中文关键词: 脑微血管内皮细胞 Necroptosis z-VAD-FMK 缺血再灌注损伤 |
| English Keywords:Brain microvascular endothelial cells Necroptosis z-VAD-FMK Ischemia-reperfusion injury |
| 基金项目:国家自然科学基金项目（编号：81273885）；北京市自然科学基金项目（编号：7144223） |
|
| 摘要点击次数: 1039 |
| 全文下载次数: 1331 |
| 中文摘要: |
| 目的：采用拟缺血再灌注损伤结合z-VAD-FMK（Benzyloxyearbonyl-Val-Ala-Asp-fluoromethylketone，z-VAD-FMK）干预，探索一种脑微血管内皮细胞（Brain Microvascular Endothelial Cells，BMECs)Necroptosis模型。方法：首先利用原代大鼠脑微血管内皮细胞，采用氧糖剥夺及复氧复糖方法，筛选出拟缺血再灌注损伤时间点。在拟缺血再灌注模型基础上，予Casepase抑制剂z-VAD-FMK 20 μmol/L干预，采用CCK-8检测细胞活性；透射电镜观察细胞超微结构；Annexin V-FITC/PI(Propidium Iodide）双染色法检测细胞死亡方式。结果：确定氧糖剥夺2 h复氧复糖8 h，作为拟缺血再灌注时间点；z-VAD-FMK作用于拟缺血再灌注损伤BMECs后，细胞活性无统计学意义；z-VAD-FMK干预组在电镜下呈现明显的Necroptosis特征；流式检测显示，各象限细胞比率无明显变化，但Necroptosis特异性抑制剂Nec-1可显著降低Q2象限细胞比率，提示z-VAD-FMK干预抑制了细胞晚期凋亡，诱导了Necroptosis的发生。结论：z-VAD-FMK可诱导拟缺血再灌注脑微血管内皮细胞发生Necroptosis，为以后研究缺血性脑中风necroptosis机制提供了细胞实验模型。 |
| English Summary: |
| To explore a model of necroptosis in brain microvascular endothelial cells (BMECs) by mimic ischemia-reperfusion injury combined with z-VAD-FMK (Benzyloxyearbonyl-Val-Ala-Asp-fluoromethylketone， z-VAD-FMK). Methods:First， primary rat BMECs were cultured in oxygen-glucose deprivation (OGD) and reintroduction conditions for various time points to screen a time of mimic ischemia-reperfusion injury. Then， z-VAD-FMK， the casepase inhibitor， was administrated at 20 μmol/L concentration on the ischemia-reperfusion injured BMECs. The cell activity was detected by CCK-8. The ultra-stucture was observed using the transmission electron microscope. The death mode of BMECs was detected using cell ultrastructure; Annexin V-FIT C/PI (propidium iodide) double staining to detect the type of cell death. Results:OGD 2 h and reintroduction 8 h was determined to be the time point of mimic ischemia-reperfusion injury. After z-VAD-FMK was administrated on ischemia-reperfusion injuried BMECs， the cells viability didn’t change significantly. The necroptosis characteristic was presented under transmission electron microscope. The flowcytometry results showed that ratio of cells in each quadrant had no significant change. However， Nec-1， a specific inhibitor of necroptosis， could significantly decrease the ratio of cells in quadrant Q2， suggesting that intervention of z-VAD-FMK inhibited the late apoptosis and induced necroptosis occurrence. Conclusion:The necroptosis may be induced by z-VAD-FMK in ischemia-reperfusion injured BMECs， which provides the cell experimental model for researching necroptosis mechanism in the ischemic stroke in the future. |
| 查看全文 查看/发表评论 下载PDF阅读器 |
