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目的通过乳鼠心肌细胞原代培养进行氧糖剥夺-氧糖恢复,诱导心肌细胞凋亡。方法取1~3天的新生SD乳鼠心室肌培养,采用氧糖剥夺2小时模拟缺血,氧糖恢复模拟再灌注,根据氧糖恢复时间,随机分为OGR 2小时组、OGR 4小时组、OGR 6小时组,正常培养为sham组。用Annexin V-FITC/PI双染色及JC-1染色流式细胞术分别检测细胞凋亡和膜电位,western blot检测细胞凋亡相关蛋白等。结果 OGR 4小时组、OGR 6小时组与sham组相比,Annexin V-FITC/PI双染色示细胞早期凋亡率均增高,差异有统计学意义(P<0.05),但OGR 6小时组细胞坏死率高于OGR 4小时组(P<0.05);与sham组比,OGR 2小时组、OGR 4小时组、OGR 6小时组绿色荧光比例均增高,差异有统计学意义(P<0.05);OGR 4小时组、OGR 6小时组与sham组比较,胞浆/线粒体cyt c比例增高,细胞裂解液caspase-3增高;OGR 6小时组与OGR 4小时组相比,胞浆/线粒体cyt c比例下降,差异有统计学意义(P<0.05)。结论乳鼠心肌细胞氧糖剥夺2小时-氧糖恢复4小时可诱导线粒体途径细胞凋亡。
Objective To induce cardiomyocyte apoptosis by oxygen glucose deprivation - oxygen sugar recovery via primary culture of neonatal cardiomyocytes. Methods Newborn SD neonatal rat ventricular myocytes were isolated from 1 to 3 days and were subjected to hypoxia-deprivation for 2 hours to simulate ischemia and reoxygenation. According to the recovery time of oxygen glucose, they were randomly divided into 2 hours OGR group, 4 hours OGR group , OGR 6 hours group, normal culture sham group. Cell apoptosis and membrane potential were detected by Annexin V-FITC / PI double staining and JC-1 staining respectively, and Western blot was used to detect apoptosis-related proteins. Results Compared with Sham group, OGR 4-hour group and OGR 6-hour group showed that the rate of early apoptosis was significantly higher than that of Sham group (P <0.05), but the percentage of cells in OGR 6-hour group The necrosis rate was higher than that of OGR for 4 hours (P <0.05). Compared with sham group, the ratio of green fluorescence in OGR 2 hours, OGR 4 hours and OGR 6 hours were significantly increased (P <0.05). OGR 4 hours group, OGR 6 hours group compared with sham group, cytosolic / mitochondrial cyt c ratio increased, cell lysate caspase-3 increased; OGR 6 hours group compared with OGR 4 hours group, cytosolic / mitochondrial cyt c ratio Decreased, the difference was statistically significant (P <0.05). Conclusion Oxygen-glucose deprivation in neonatal rat cardiomyocytes for 2 hours - recovery of oxygen sugar for 4 hours induces mitochondrial pathway apoptosis.