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为探讨p53上调凋亡调制物(p53 up-regulated modulator of apoptosis,PUMA)在大鼠心肌细胞缺氧/复氧(hypoxia/reoxygenatio,H/R)损伤中的作用,本研究将靶向PUMA的siRNA(si-PUMA)转染大鼠心肌细胞以建立PUMA沉默表达模型,观察其对心肌细胞H/R损伤的影响.RT-PCR和Western印迹结果表明,最适转染浓度50 nmol/L si-PUMA能靶向抑制H/R损伤心肌细胞的PUMA表达;MTT法检测心肌细胞存活率及培养基乳酸脱氢酶(lactate dehydrogenase,LDH)活性测定结果发现,si-PUMA组细胞存活率较H/R 6 h模型组明显提高,培养液中LDH活性显著降低(P<0.01);分光光度法及Annexin V-FITC/PI联合染色流式细胞凋亡检测结果显示,si-PUMA组caspase-3活性较H/R 6h组明显下调,细胞凋亡率明显降低(P<0.01);RT-PCR结果提示,与H/R6 h组相比,si-PUMA组Bax及Bcl-2表达分别出现显著下调及上调(P<0.05).以上结果表明,靶向PUMA的siRNA转染能明显增强心肌细胞耐受H/R损伤的能力,对心肌细胞具有较好的保护作用;PUMA介导H/R诱导的心肌细胞凋亡,是心肌缺血/再灌注损伤基因治疗的一个潜在靶点.
In order to investigate the role of p53 up-regulated modulator of apoptosis (PUMA) in hypoxia / reoxygenation (H / R) injury in rat cardiomyocytes, siRNA (si-PUMA) was transfected into rat cardiomyocytes to establish PUMA silence expression model and observe its effect on myocardial H / R injury.RT-PCR and Western blotting results showed that the optimal transfection concentration of 50 nmol / L si -PUMA could inhibit the PUMA expression in H / R-injured myocardial cells. The viability of cardiomyocytes and the activity of lactate dehydrogenase (LDH) were measured by MTT assay. The survival rate of cells in si-PUMA group was higher than that of H (P <0.01). The results of spectrophotometry and Annexin V-FITC / PI combined with flow cytometry showed that the expression of caspase-3 in si-PUMA group was significantly increased Compared with H / R6h group, the expression of Bax and Bcl-2 in H-R group was significantly lower than that in H / R 6h group (P <0.01). The results of RT-PCR indicated that the expression of Bax and Bcl- Downregulation and upregulation (P <0.05) .This result indicated that siRNA transfection targeting PUMA significantly enhanced the ability of cardiomyocytes to tolerate H / R injury, Cardiomyocytes has good protective effect; PUMA mediated H / R-induced apoptosis of cardiomyocytes, myocardial ischemia / reperfusion injury in a potential target for gene therapy.