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目的探讨Ca2+预处理对未成熟心肌细胞凋亡和凋亡相关蛋白的影响。方法实验于2000年6月至2001年12月在武汉大学人民医院心血管外科完成。日本长耳大白兔12只,雌雄不限,14~21 d,体重230~300 g。按随机数字表法分为2组,每组6只。缺血-再灌注组(I/R组):常规建立Langendorff模型,灌注重碳酸氢盐缓冲液(Krebs-Henseleit,KH)20 min,停灌45 min,复灌120 min;钙预处理组(CP组):建立Langendorff模型,灌注KH液20 min后无钙KH液灌注45 s,反复3次,KH液灌注5 min,停灌45 min,复灌120 min。采用凋亡细胞原位标记和半定量分析检测心肌细胞凋亡率,琼脂糖凝胶电泳检测DNA片段梯,Western blot法检测bcl-2、bax和fas的表达。结果 CP组的心肌细胞凋亡率低于I/R组(4.53%±1.22%vs.12.30%±2.12%,t=7.780,P=0.000)。CP组琼脂糖凝胶电泳检测的DNA片段梯与I/R组比较明显减弱(光密度值:56460±1640 vs.135212±3 370,t=51.460,P=0.000)。I/R组bcl-2表达与CP组比较明显减少(光密度值:13217±1 770 vs.31790±1 018,t=22.280,P=0.000),I/R组bax(光密度值:30 176±1025 vs.7954±730,t=43.260,P=0.000)和fas(光密度值:29 197±1 233vs.8140±867,t=34.220,P=0.000)表达与CP组比较均明显增高。结论 Ca2+预处理能影响bcl-2、bax和fas的表达,可减少未成熟心肌细胞凋亡。
Objective To investigate the effect of Ca2 + pretreatment on immature myocardial cell apoptosis and apoptosis related proteins. Methods The experiment was performed in Cardiovascular Surgery of People’s Hospital of Wuhan University from June 2000 to December 2001. Japanese long-eared rabbits 12, male or female, 14 ~ 21 d, weight 230 ~ 300 g. According to random number table divided into 2 groups, each group 6. The ischemia-reperfusion group (I / R group): Langendorff model was established routinely, and Krebs-Henseleit (KH) was infused for 20 min, CP group): Langendorff model was established. After perfusion of KH solution for 20 min, calcium-free KH solution was perfused for 45 s and repeated 3 times. KH solution was infused for 5 min. Apoptotic rate of cardiomyocytes was detected by in situ and semi-quantitative analysis of apoptotic cells. The DNA ladder was detected by agarose gel electrophoresis and the expression of bcl-2, bax and fas was detected by Western blot. Results The apoptosis rate of cardiomyocytes in CP group was lower than that in I / R group (4.53% ± 1.22% vs.12.30% ± 2.12%, t = 7.780, P = 0.000). The DNA fragment ladder detected by agarose gel electrophoresis in CP group was significantly weaker than that in I / R group (optical density value: 56460 ± 1640 vs.135212 ± 3 370, t = 51.460, P = 0.000). The expression of bcl-2 in I / R group was significantly lower than that in CP group (optical density value: 13217 ± 1770 vs.31790 ± 1 018, t = 22.280, P = 0.000) 176 ± 1025vs.7954 ± 730, t = 43.260, P = 0.000) and fas (optical density value: 29 197 ± 1 233vs.8140 ± 867, t = 34.220, P = 0.000) . Conclusion Ca2 + preconditioning can affect the expression of bcl-2, bax and fas and reduce the apoptosis of immature cardiomyocytes.