PUMA介导大鼠缺氧/复氧心肌细胞凋亡的研究  

Study on PUMA-mediated hypoxia-reoxygenation-induced rat cardiomyocyte apoptosis

作　　者：付晶晶[1,2] 段荣[2] 李红[2] 万福生[1] 

机构地区：[1]南昌大学基础医学院生物化学与分子生物学教研室,江西南昌330006 [2]江西省儿童医院检验科,江西南昌330006

出　　处：《中国药理学通报》2011年第3期312-316,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No30060029);江西省自然科学基金资助项目(No2010JZY0237)

摘　　要：目的研究促凋亡蛋白p53上调凋亡调制物(PUMA)在大鼠缺氧/复氧(H/R)心肌细胞凋亡中的作用及意义。方法原代培养的大鼠心肌细胞缺氧5 h后复氧2～12 h以制备缺氧/复氧损伤模型,靶向PUMA的siRNA转染大鼠心肌细胞以建立PUMA沉默表达模型。采用Annexin V-FITC联合PI双染流式细胞术检测细胞凋亡率;分光光度法检测Caspase-3活性变化;RT-PCR、Western blot等方法分析PUMA及凋亡相关基因Bax、Bcl-2的mRNA及蛋白表达变化。结果与正常对照组相比,H/R处理后心肌细胞凋亡率及Caspase-3活性迅速上升;PUMA mRNA及蛋白表达上调,凋亡相关蛋白Bax表达上调及Bcl-2的表达下调。靶向PUMA的siRNA转染后,Bax上调表达及Bcl-2下调表达的程度明显被抑制。结论在大鼠心肌细胞缺氧/复氧过程中,PU-MA表达明显升高,其可能通过下调Bcl-2表达及上调Bax表达导致Caspase-3活性增加以介导心肌细胞凋亡。Aim To investigate the effect of PUMA in hypoxia-reoxygenation（H/R）induced cardiomyocyte apoptosis.Methods Primary cultured rat cardiomyocytes were exposed to hypoxia for 5 hours followed by reoxygenation for 2~12 hours.PUMA targeted siRNA was transfected into primary cultured cardiomyocytes in vitro.Cell apoptosis was determined by Annexin V apoptosis assay and PI staining combined Flow Cytometry.Spectrophotometer was used to test caspase-3 activity.RT-PCR and Western blot were used to semi-quantify the mRNA and protein of PUMA,Bcl-2 and Bax,respectively.Results In comparison with the control group,cell apoptosis and Caspase-3 activity of the H/R group were increased.PUMA,Bax expression was up-regulated while Bcl-2 was down-regulated upon H/R treatment.Moreover,it was found that PUMA targeted siRNA could inhibit the effect by neutralizing Bax and Bcl-2 expression changes.Conclusion PUMA is induced significantly in hypoxia-reoxygenation rat cardiomyocytes,which may mediate apoptosis through upregulation of Bax and downregulation of Bcl-2.

关 键 词：p53上调凋亡调制物 BH3-only蛋白 缺血/再灌注损伤 缺氧/复氧 心肌细胞 凋亡 小干扰RNA 

分 类 号：R-332[医药卫生] R322.11