大鼠肾小管上皮细胞缺血后处理模型的建立

Establishment of an in vitro model system for investigating ischemic postconditioning using renal tubular epithelial cell of rats

目的探讨用大鼠肾小管上皮细胞NRK-52E建立一种可以用于缺血后处理研究的体外模型,并找到最佳的后处理方案。方法将NRK-52E随机分为4组:对照组(Sham),缺血再灌注(IRI)组,换液缺血后处理(HP)组,加液缺血后处理(HP+)组。通过体外糖氧剥夺3 h后模拟缺血再灌注损伤。通过行循环10 min再灌注/10 min缺血,建立肾缺血后处理的体外模型。流式细胞仪用于检测细胞凋亡。蛋白印迹法检测细胞质内细胞色素C蛋白水平。结果 NRK-52E经过细胞缺血再灌注24 h后有明显凋亡(P<0.05)。部分后处理组凋亡明显减轻(P<0.05),其中以HP+组保护效果最佳。NRK-52经过细胞缺血再灌注24 h后细胞质内细胞色素C蛋白的表达都明显升高(P<0.05),缺血后处理组细胞相应的蛋白表达显著减轻,其中以HP+最为显著。结论成功建立NRK-52E缺血后处理的体外模型,其机制可能与通过抑制凋亡有关。

[ Objective ] To establish an in vitro model system for investigating ischemic postconditioning using renal tubular epitheli- al cell NRK-52E of rats, study the optimal scheme for ischemic postconditioning. [ Methods] NRK-52E ceils were randomly divided into 4 groups ： Sham group, IRI group, HP group, and HP ＋ group. In order to establish the in vitro model of renal ischemic post- conditioning, the cells were treated with oxygen-glucose deprivation for 3 hours to simulate the？ ischemia-reperfusion injury, and then the cells were exposed to the cycles of reperfusion condition for 10 minutes and ischemic condition for 10 minutes. The flow cy- tometry was used to test apoptosis, and the Western blotting technique was adopted to evaluate the level of cytochrome C. [ Results] There was obvious apoptosis in NRK-52E cells subjected to ischemia-reperfusion for 24 hours （ P 〈 0. 05 ）. The apoptosis in some ischemic postconditioning groups reduced significantly （P 〈 0.05 } , and the protection effect in HP ＋ group was the best. The ex- pression of cytochrome C increased significantly in NRK-52E cells subjected to ischemia-reperfusion for 24 hours （ P 〈 0. 05 ）. In contrast, the expression of cytochrome C in ischemic postconditioning groups attenuated significantly, especially HP ＋ group. [ Conclusion] The in vitro model of ischemic postconditioning of NRK-52E is established successfully, and its mechanism？ may be related to inhibition of apoptosis.