大鼠心肌细胞凋亡的保护作用

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/R 6 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.

11PUMA-targeting siRNA Protects Rat Cardiomyocyte from Hypoxia/Reoxygenation-induced Apoptosis

To study the effect of p53 up-regulated modulator of apoptosis (PUMA) in hypoxia/ reoxygenation (H/R) mediated rat cardiomyocyte injury, siRNA targeted PUMA was designed and transfected into primary cultured rat cardiomyocytes in parallel with a scramble siRNA. Optimized dose of PUMA specific siRNA was 50 nmol/L as determined by RT-PCR and Western blotting. The MTT assay and lactate dehydrogenase (LDH) activity measurement showed that the cell survival was enhanced and LDH release was reduced after the transfection of PUMA siRNA (P<0.01, compared with the control). PUMA siRNA could also down-regulate caspase-3 activity and decrease apoptosis rate (P<0.01) from the results of annexin V apoptosis assay and PI-stained with flow cytometry. Down-regulation of Bax and up-regulation of Bcl-2 were detected in RT-PCR (P<0.05). These results indicated that PUMA siRNA could protect cardiomyocytes from apoptosis and could be considered for the therapy for cardiac ischemia/reperfusion injury.