Tu Shuo, Liu Zhuo-Qi, Fu Jing-Jing, Zhu Wei-Feng, Luo Da-Ya, Wan Fu-Sheng
Department of Biochemistry and Molecular Biology, Basic Medical College of Nanchang University, Nanchang, China.
Cardiology. 2012;122(2):93-100. doi: 10.1159/000338701. Epub 2012 Jun 30.
The effect of p53 upregulated modulator of apoptosis (PUMA) in hypoxia/reoxygenation-induced cardiomyocyte injuries in rats was investigated.
PUMA-targeting (si-PUMA) and scramble siRNAs were designed and transfected into primarily rat cardiomyocytes in vitro.
RT-PCR and Western blot analysis showed that 50 nmol/l of si-PUMA can specifically inhibit PUMA expression. MTT assay and lactate dehydrogenase activity detection showed that the cell survival rate in the si-PUMA group was enhanced and that the lactate dehydrogenase enzymatic activity dramatically decreased compared with the control group (p < 0.01). Spectrophotometry, as well as annexin V and propidium iodide staining, combined with flow cytometry, revealed that caspase-3 activity in the si-PUMA group was downregulated and the apoptotic rate was decreased (p < 0.01). RT-PCR also showed that Bax expression was downregulated and Bcl-2 expression was upregulated in the si-PUMA group, compared with the control group (p < 0.05). si-PUMA protects cardiomyocytes from apoptosis.
PUMA mediates hypoxia/reoxygenation-induced cardiomyocyte apoptosis, which can be a potential target of gene therapy for ischemia/reperfusion cardiomyocyte injuries.
研究p53上调凋亡调节因子（PUMA）在大鼠缺氧/复氧诱导的心肌细胞损伤中的作用。
设计针对PUMA的（si-PUMA）和乱序siRNA，并在体外原代大鼠心肌细胞中进行转染。
RT-PCR和蛋白质印迹分析表明，50 nmol/l的si-PUMA可特异性抑制PUMA表达。MTT法和乳酸脱氢酶活性检测显示，与对照组相比，si-PUMA组细胞存活率提高，乳酸脱氢酶活性显著降低（p < 0.01）。分光光度法以及膜联蛋白V和碘化丙啶染色结合流式细胞术显示，si-PUMA组caspase-3活性下调，凋亡率降低（p < 0.01）。RT-PCR还显示，与对照组相比，si-PUMA组Bax表达下调，Bcl-2表达上调（p < 0.05）。si-PUMA可保护心肌细胞免于凋亡。
PUMA介导缺氧/复氧诱导的心肌细胞凋亡，这可能是缺血/再灌注心肌细胞损伤基因治疗的潜在靶点。
Zotero 插件