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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 of rat renal tubular epithelial cells NRK-52E that can be used for post-ischemic postconditioning and to find the best post-treatment regimen. Methods NRK-52E was randomly divided into 4 groups: control group (Sham), ischemia-reperfusion group (IRI), replacement fluid ischemic postconditioning (HP) and ischemic postconditioning (HP +). Simulated ischemia-reperfusion injury after 3-hour Oxygen deprivation in vitro. In vitro model of postischemic renal ischemia was established by reperfusion of 10 min ischemia / reperfusion for 10 min. Flow cytometry was used to detect apoptosis. Cytochrome C protein levels in the cytoplasm were detected by Western blotting. Results NRK-52E showed obvious apoptosis after 24 h reperfusion (P <0.05). Part of the post-treatment group apoptosis was significantly reduced (P <0.05), which HP + group to protect the best. The expression of cytochrome C protein in the cytoplasm of NRK-52 cells was significantly increased after 24 h of ischemia reperfusion (P <0.05), and the protein expression of NRK-52 in the ischemic postconditioning group was significantly reduced, with HP + being the most significant. Conclusion The successful establishment of NRK-52E ischemic postconditioning in vitro model may be related to the inhibition of apoptosis.