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目的以细胞凋亡的线粒体途径为切入点,探讨1类多巴胺受体(DR1)活性变化对缺氧、复氧诱导的心肌细胞凋亡的影响及可能机制。方法乳鼠心肌细胞以缺氧、缺血清培养2小时,复氧、复血清培养24小时的方法建立心肌细胞缺氧、复氧损伤模型。RT-PCR和Western blotting分别检测Bcl-2、caspase-3、caspase-9和细胞色素C(Cyt c)mRNA和蛋白质的表达;TUNEL、流式细胞仪、MTT检测心肌细胞的凋亡情况;紫外分光光度计检测细胞培养液中LDH、SOD活性和MDA含量;透射电镜观察心肌细胞形态变化。结果与正常组比较,缺氧-复氧组细胞培养液中LDH活性和MDA含量增加,SOD活性降低;心肌细胞凋亡指数增加;心肌细胞超微结构损伤加重;促凋亡因子(Cyt c、caspase-3、caspase-9)表达增加,抑凋亡因子(Bcl-2)表达亦增加。与缺氧-复氧组比较,DR1激动剂SKF-38393进一步增加LDH活性和MDA含量,降低SOD活性,增加心肌细胞凋亡指数,加重心肌细胞损伤,上调促凋亡因子表达,下调抑凋亡因子表达;DR1抑制剂SCH-23390对上述指标影响不明显。结论 DR1激活可促进缺氧、复氧诱导的心肌细胞凋亡,其机制与上调线粒体途径有关。
Objective To investigate the effects of the change of dopamine receptor (DR1) activity on the apoptosis of cardiomyocytes induced by hypoxia and reoxygenation through the mitochondrial pathway of apoptosis. Methods Cardiomyocytes were exposed to hypoxia and ischemia for 2 hours, reoxygenated and incubated with serum for 24 hours to establish hypoxia and reoxygenation injury models. The expression of Bcl-2, caspase-3, caspase-9 and Cyt c mRNA and protein were detected by RT-PCR and Western blotting respectively. The apoptosis of cardiomyocytes was detected by TUNEL, flow cytometry and MTT. The LDH, SOD activity and MDA content in cell culture medium were detected by spectrophotometer. The morphological changes of myocardial cells were observed by transmission electron microscope. Results Compared with the normal group, the activities of LDH and MDA in the hypoxia-reoxygenation group increased, the activity of SOD decreased, the apoptosis index of cardiomyocytes increased, the ultrastructural damage of cardiomyocytes increased, the expression of Cyt c, caspase-3, caspase-9) increased, while the expression of apoptosis-inhibiting factor (Bcl-2) also increased. Compared with hypoxia-reoxygenation group, DR1 agonist SKF-38393 further increased LDH activity and MDA content, decreased SOD activity, increased cardiomyocyte apoptosis index, increased cardiomyocyte injury, increased pro-apoptotic factor expression, and down-regulated apoptosis Factor expression; DR1 inhibitor SCH-23390 had no significant effect on the above indicators. Conclusion DR1 activation can promote hypoxia and reoxygenation-induced cardiomyocyte apoptosis, the mechanism of which is related to up-regulation of mitochondria pathway.