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目的以细胞凋亡的死亡受体(Fas/Fas-L)途径为切入点,探讨1类多巴胺受体(dopamine receptor-1,DR1)活性变化对缺血-再灌注损伤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模拟缺血溶液培养2 h后再正常培养24 h的方法建立心肌细胞缺血-再灌注损伤模型。心肌细胞随机分为4组:正常对照组,缺血-再灌注组(I/R组),10μmol/L SKF-38393(DR1激动剂)组(SKF干预组),10μmol/L SCH-23390(DR1抑制剂)组(SCH干预组)。RT-PCR和Western blot分别检测Fas、Fas-L、Caspase-8、Caspase-3 mR-NA和蛋白质的表达;TUNEL染色和流式细胞仪技术观察心肌细胞的凋亡情况;紫外分光光度计检测细胞培养液中乳酸脱氢酶(lactate dehydrogenase,LDH)、超氧化物歧化酶(superoxide dismutase,SOD)活性和丙二醛(malondialdehyde,MDA)含量;透射电镜观察心肌细胞形态变化。结果与正常对照组比较,I/R组中Fas、Fas-L、caspase-8、caspase-3表达均上调,LDH活性和MDA含量增加,而SOD活性降低,心肌细胞损伤加重;与I/R组比较,10μmol/L SKF-38393使上述指标进一步增加,而10μmol/L SCH-23390对上述指标影响不显著。结论 DR1激活能够促进缺血-再灌注损伤诱导的细胞凋亡,其机制与上调Fas/Fas-L死亡途径有关。
Objective To investigate the effect of dopamine receptor-1 (DR1) activity on apoptosis of cardiomyocytes induced by ischemia-reperfusion (I / R) injury by using the apoptotic death receptor (Fas / Fas-L) Impact and possible mechanisms. Methods Cardiomyocytes were isolated from neonatal rat cardiomyocytes for 2 hours and then cultured for 24 hours. The cardiomyocyte ischemia-reperfusion injury model was established. Cardiomyocytes were randomly divided into 4 groups: normal control group, ischemia / reperfusion group (I / R group), 10μmol / L SKF-38393 (DR1 agonist group) DR1 inhibitor) group (SCH intervention group). The expression of Fas, Fas-L, Caspase-8 and Caspase-3 mRNA and protein were detected by RT-PCR and Western blot respectively. The apoptosis of cardiomyocytes was observed by TUNEL staining and flow cytometry. The activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the cell culture medium were observed. The morphological changes of myocardial cells were observed by transmission electron microscope. Results Compared with the normal control group, the expression of Fas, Fas-L, caspase-8 and caspase-3 in I / R group were increased, the activity of LDH and the content of MDA were increased, while the activity of SOD was decreased and the injury of cardiomyocytes was increased. Compared with the control group, 10μmol / L SKF-38393 further increased the above parameters, while 10μmol / L SCH-23390 had no significant effect on these indexes. Conclusion Activation of DR1 can promote apoptosis induced by ischemia-reperfusion injury. Its mechanism is related to the up-regulation of Fas / Fas-L death pathway.