过表达肌浆网钙ATP酶对心肌细胞内质网应激的影响

目的 研究过表达肌浆网钙ATP酶2a(SERCA2a)对心肌细胞内质网应激的影响.方法 构建含SERCA2a cDNA的重组腺病毒载体(Adv-SERCA2a),以缺氧和衣霉素诱导内质网应激,随机将体外培养的心肌细胞分为以下6组:正常对照组、缺氧组、衣霉素组、过表达SERCA2a组、SERCA2a+缺氧组和SERCA2a+衣霉素组.采用Western印迹法检测内质网应激蛋白GRP78及C/EBP同源蛋白(CHOP)表达水平,流式细胞术检测心肌细胞凋亡率,台盼蓝染色检测心肌细胞存活率.结果 过表达SERCA2a+缺氧组GRP78及CHOP蛋白表达较缺氧组分别低49.1%和52.7%,细胞凋亡率较缺氧组低66.0%,细胞存活率较缺氧组高13.4%,差异有统计学意义(均P<0.05);过表达SERCA2a+衣霉素组GRP78及CHOP蛋白表达较衣霉素组分别低50.4%和66.1%,细胞凋亡率较衣霉素组低54.0%,细胞存活率较衣霉素组高6.7%,差异有统计学意义(均P<0.05).结论 过表达SERCA2a通过下调内质网应激蛋白表达,降低心肌细胞内质网应激水平,缓解过度内质网应激介导的细胞损伤.

Effects of sarcoplasmic reticulum calcium ATPase 2a overexpression on endoplasmic reticulum stress in cardiomyocytes

Objective To investigate the effects of sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) overexpression on the endoplasmic reticulum stress (ERS) . Methods Ventricular cardiomyocytes were obtained from a neonatal rat,cultured,and then randomly divided into 6 groups: normal control group;hypoxia group cultured in an airtight chamber gassed with 95% N2/5% CO2 at 37℃ for 72 hours so as to induce ERS;tunicamyein group treated with 10 μg/ml tunicamycin so as to induce ElLS too;SERCA2a group transfected with recombinant adenovirus expressing the target gene SERCA2a (rAd-SERCA2a) ;SERCA2a+hypoxia group;and SERCA2a+tunicamycin group. Western blotting was used to detec the protein expression of SERCA2a,and glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) ,both the ERS markers. Flow cytometry was used to detect the apoptosis of the cardiomyocytes,and trypan blue staining was used to detect the survival rate of the cardiomyocytes. Results (1) The GRP78 expression level of the hypoxia group was 5.1 times as high as that of the control group,and the CHOP expression level of the hypoxia group was 2.5 times as high as hat of the control group. The GRP78 expression level of the tunicamycin group was 4.9 times as high as that of the control group,and the CHOP expression level of the tunicamycin group was 3.1 times as high as hat of the control group. SERCA2a overexpression was found to relieve the expression of GRP78 induced by hpoxia and tunicamycin (49.1% and 50.4% decrease respectively),and to inhibit the activation of CHOP (52.7% and 66.1% decrease respectively). (2) In comparison with the hypoxia group,the protein expression levels of GRP78 and CHOP of the SERCA2a overexpression + hypoxia group were significantly lower by 49.1% and 52.7% respectively,the apeptotic rate was significantly lower by 66.0%,and the cardiomyocyte survival rate was significantly higher by 13.4% (all P < 0.05). Compared with the tunicamycin group,the protein expression levels of GRP78 and CHOP of the SERCA2a overexpression + tunicamycin group were significantly lower by 50.4% and 66.1% respectively,the apoptotic rate was significantly lower by 54.0%,and the cadiomyocyte survival rate was significantly higher by 6.7% (all P < 0.05). Conclusion SERCA2a overexpression attenuates ERS induced by hypoxia or tunicamycin,and protects cardiomyocytes against ERS-mediated cellular injury.