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目的 观察肿瘤坏死因子(TNF-α)诱导的离体血管内皮细胞损伤后线粒体功能、过氧化物酶体增殖物活化受体γ共激活因子-1α(PGC-1α)、活化T细胞核因子1(NFAT1)、活化T细胞核因子2(NFAT2)的变化.方法 (1)不同浓度的TNF-α(10、20、40、80 ng/mL)刺激人脐静脉内皮细胞(HUVECs)12、24、36 h制备不同程度的血管内皮细胞损伤模型,CCK-8法检测细胞活性、AnnexinⅤ-FITC/PI双染法检测细胞凋亡,倒置光学显微镜下观察细胞形态,以此确定模型是否建立成功.(2)采用Jc-1免疫荧光染色结合荧光显微镜检测线粒体膜电位,MitoSOX?Red染色结合荧光显微镜检测线粒体活性氧(mtROS).(3)采用RT-qPCR、Western blot方法分别检测PGC-1α、NFAT1、NFAT2 mRNA及蛋白水平.结果 (1)随着TNF-α刺激浓度及作用时间的增加,HUVECs活性逐渐下降,在浓度为40 ng/mL,刺激时间为24 h,细胞活性下降约49％,接近TNF-α诱导HUVECs损伤的IC50,差异有统计学意义(P<0.05).20、40、80 ng/mL TNF-α刺激HUVECs 24 h后,细胞凋亡率逐渐升高,差异具有统计学意义(P<0.05).(2)随着血管内皮细胞损伤程度的增加,线粒体膜电位逐渐下降,mtROS逐渐升高(均P<0.05).(3)PGC-1αmRNA及蛋白表达水平逐渐下降,核转录因子NFAT1、NFAT2 mRNA及蛋白表达水平逐渐升高(均P<0.05).结论 随着血管内皮细胞损伤程度的增加,线粒体功能障碍逐渐加剧,NFAT表达量逐渐增加.“,”Objective To observe the changes of mitochondrial function, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and nuclear factor of activated T cell (NFAT) in TNF-α-induced injury of vascular endothelial cells. Methods (1)Different concentrations of tumor necrosis factor TNF-α (10, 20, 40, 80 ng/mL) stimulated human umbilical vein endothelial cells (HUVECs) 12, 24, 36 hours to prepare different degrees of vascular endothelial cell injury model, and CCK-8 method was used to detect the cell viability. Apoptosis was detected by AnnexinⅤ-FITC/PI double staining method. Morphological changes were observed under inverted optical microscope to determine whether the model was established successfully. (2)The mitochondrial membrane potential was detected by JC-1 immunofluorescence staining and fluorescence microscopy, MitoSOX? Red staining combined with fluorescence microscopy was used to detect mitochondrial reactive oxygen species. (3)RT-qPCR, Western blot method to detect mRNA and protein levels of PGC-1α, NFAT1 and NFAT2. Results (1)With the increase of TNF-α concentration and the time of action, the activity of HUVECs decreased gradually. When the concentration was 40 ng/mL and the stimulation time was 24 hours, the cell viability decreased by about 49％, which was close to the IC50 of HUVECs injury induced by TNF-α. The difference was statistically significant (P<0.05). After treatment with TNF-α at the concentration of 20, 40 and 80 ng/mL, the apoptotic rate of HUVECs increased gradually, and the difference was statistically significant (P<0.05). (2)With the increase of vascular endothelial cell injury, the mitochondrial membrane potential was decreased gradually, and the level of mtROS was increased gradually (all P<0.05). (3)The mRNA and protein levels of PGC-1α were decreased gradually, and the mRNA and protein levels of NFAT1, NFAT2 were increased gradually (all P<0.05).Conclusion With the increase of vascular endothelial cell injury, mitochondrial dysfunction gradually increased, NFAT expression increased gradually.