缺氧后处理和二氮嗪后处理对大鼠心肌细胞缺氧复氧时钙网蛋白表达的影响

Effect of hypoxic postconditioning and diazoxide postconditioning on calreticulin expression during hypoxia/ reoxygenation in rat cardiomyocytes

目的 探讨缺氧后处理和二氮嗪后处理对大鼠心肌细胞缺氧复氧时钙网蛋白（CRT）表达的影响.方法 健康雄性SD大鼠,4～5月龄,分离心肌细胞,培养18 h后,随机分为6组（n=8）：对照组（C组）、缺氧复氧组（HR组）、缺氧后处理组（HP组）和二氮嗪后处理组（DP组）.C组细胞于5%CO2培养箱内继续培养2 h;HR组细胞于95%N2-5%CO2培养箱中缺氧45 min,然后于95%O2-5%CO2培养箱中复氧1 h;HP组细胞于95%N2-5%CO2培养箱中缺氧45 min,然后复氧3 min,缺氧3 min,重复3次,再于95%O2-5%CO2培养箱中复氧1 h;DP组细胞于95%N2-5%CO2培养箱中缺氧45 min,然后给予50 μmol/L二氮嗪处理10 min,再于95%O2-5%CO2培养箱中复氧1 h.测定心肌细胞caspase-3活性、CRT表达和游离钙离子浓度.结果 与C组比较,其余各组caspase-3活性升高,HP组和DP组CRT表达上调,HR组游离钙离子浓度升高（P＜0.05或0.01）;与HR组比较,HP组和DP组caspase-3活性降低,CRT表达上调,游离钙离子浓度降低（P＜0.01）.结论 缺氧后处理和二氮嗪后处理可上调CRT的表达,减轻细胞内钙超载,减轻大鼠心肌细胞缺氧复氧损伤.

Objective To investigate the effect of hypoxic postconditioning and diazoxide postconditioning on the calreticulin expression during hypoxia/reoxygenation （H/R） in rat cardiomyocytes. Methods Primary cultured male SD rat cardiomyocytes were randomly divided into 6 groups （n = 8 each）： control group （group C）,H/R group; hypoxic postconditioning group （group HP） and diazoxide postconditioning group （group DP）. Group C were cultured continuously for 2 h. Group H/R, Hp and DP were exposed to 45 min hypoxia （95% N2-5% CO2）followed by 1 h reoxygenation. In group HP, the cells were subjected to three cycles of 3 min hypoxia at 3 min intervals at the end of 45 min hypoxia before 1 h reoxygenation. In group DP, diazoxide 50 μmol/L was giyen at the end of hypoxia. Caspase-3 activity, calreticulin expression and intracellular free calcium ion concentrations were determined. Results Compared with group C, caspase-3 activity was significantly increased in the other groups,the calreticulin expression was up-regulated in group HP and DP, and the free calcium ion concentrations were increased in group H/R （P 〈 0.05 or 0.01）. Compared with group H/R, caspase-3 activity was significantly decreased in HP and DP, the calreticulin expression was up-regulated and the free calcium ion concentrations were decreased in group HP and DP （P 〈 0.01）. Conclusion Hypoxic postconditioning and diazoxide postconditioning attenuate H/R injury in rat cardiomyocytes through up-regulating the expression of calreticulin and reducing intracellular calcium overload.