内质网应激在Bim介导缺氧致心肌细胞凋亡中的作用

Role of endoplasmic reticulum stress in Bim-mediated cardiomyocyte apoptosis induced by hypoxia

目的：探讨内质网应激在Bim介导缺氧致心肌细胞凋亡中的作用.方法：在体外原代培养出生1～3 d大鼠心肌细胞,并用抗α-横纹肌肌动蛋白免疫组化法进行鉴定.设计并化学合成3对靶向bim的siRNA,用脂质体法将siRNA转染心肌细胞,筛选沉默效率最高的siRNA.实验分组：（1）空白对照组;（2）缺氧组;（3）缺氧＋脂质体组;（4）缺氧＋阴性对照siRNA组;（5）缺氧＋Bim-siRNA组.MTT法观察细胞活性;流式细胞术检测细胞凋亡率及细胞内钙离子浓度变化情况;Western blotting检测内质网应激标志分子caspase-12和三磷酸肌醇（IP3）的表达情况.结果：免疫组化鉴定证实大鼠心肌细胞原代培养成功.在荧光显微镜下,转染了阴性对照siRNA组的细胞中观察到绿色荧光,即转染成功;Western blotting 结果显示,Bim-siRNA转染均能有效降低Bim蛋白的表达,其中第2对沉默效率最高,达到86.73%.缺氧损伤导致心肌细胞活性明显下降（P〈0.05）,转染Bim-siRNA后细胞活性较阴性对照组升高.缺氧细胞凋亡率较对照组明显增加（P〈0.01）,细胞内钙离子浓度明显增高,而沉默bim的表达能降低细胞凋亡率和细胞内钙离子浓度.缺氧导致内质网应激标志分子caspase-12和IP3表达较空白对照组明显上调（均P〈0.05）,而抑制Bim表达后caspase-12和IP3表达明显降低.结论：沉默bim的表达能有效抑制缺氧导致心肌细胞凋亡的作用,内质网应激标志分子caspase-12和IP3可能参与了Bim介导缺氧致心肌细胞凋亡的过程.这有望为临床心肌缺血缺氧损伤的治疗提供新思路.

AIM ： To investigate the role of endoplasmic reticulum stress （ERS） in the process of Bim-media- ted cardiomyocyte apoptosis induced by hypoxia. METHODS： Cardiomyocytes were isolated from neonatal Sprague-Dawley rats aged 1 - 3 days, and primarily cultured in vitro. The antibody targeting or-striated muscle actin was used to identify the cardiomyocytes. The siRNAs targeting bim were transfected into cardiomyocytes with liposome, followed by detecting the expression of Bim by Western blotting. Cardiomyocytes were divided into five groups ： blank control group, hypoxia group, hypoxia ＋ liposome group, hypoxia ＋ negative control siRNA group and hypoxia ＋ Bim-siRNA group. The cell viability was determined by MTr assay, and the cell apoptotic rate and the intraceUular calcium concentration were measured by flow cy- tometry. The protein expression of caspase-12 and inositol 1,4,5-triphosphate （IP3） was detected by Western blotting. RESULTS ： identification confirmed that rat cardiomyocytes were successfully cuhured. Green fluo- rescence was observed in the cells transfected with negative control siRNA under fluorescence microscope. The expression of Bim was obviously inhibited after transfected with Bim-siRNAs and the silencing efficiency of Bim-siRNA-2 was the high- est （86.73%）. Compared with blank control group, the viability of cardiomyocytes in hypoxia group was significantly re- duced （P 〈 0. 05）. Compared with hypoxia ＋ negative control siRNA group, the viability of cardiomyocytes in hypoxia ＋Bim-siRNA group was significantly increased （ P 〈 0. 05 ）. The apoptotic rate and the intracellular calcium concentration of cardiomyocytes were obviously increased .in hypoxia group （ P 〈 0.01 ）, and were both decreased after bim silencing （ P 〈 0. 05 or P 〈0.01 ）. The expression of caspase-12 and IP3 was up-regulated in hypoxia group （P 〈0.05）, and was down- regulated after bim silencing （P 〈 0.05 or P 〈0.01 ）. CONCLUSION： Cardiomyocyte apoptosis induced by hypoxia can be inhibited by silencing the expression of bim gene. Caspase-12 and IP3, as markers of ERS, may participate in the process of Bim-mediated cardiomyocyte apoptosis induced by hypoxia.