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目的探讨缺血后处理对猪心肌细胞Fas基因蛋白表达及Caspase-3活性的影响。方法24只小型约克猪(体重35～40kg)被随机分为4组。组1(ACC组,n=6)：于CPB开始后并行循环45 min,阻断主动脉90 min,开放主动脉后心脏再灌注120 min；组2(pre-con组,n=6)：升主动脉阻断前进行心脏缺血预处理(阻断升主动脉5 min、开放10 min,重复3次),余处理与组1相同；组3(post-con组,n=6)：并行循环45 min,阻断主动脉90 min,开放主动脉后心脏再灌注120 min；再灌注开始进行缺血后处理(阻断升主动脉30 s后开放30 s,重复3次,共3 min)；组4(pre-con +post-con组,n=6)：升主动脉阻断前进行心脏缺血预处理(阻断升主动脉5 min、开放10 min,重复3次),阻断主动脉90 min,开放主动脉心脏再灌注120 min,再灌注开始进行心肌缺血后处理(阻断升主动脉30 s、开放30 s,重复3次共3 min)。在再灌注结束后取左心室全层心肌适量并固定。用原位化学法(TUNEL)观察各组心肌细胞凋亡,流式细胞法检测Fas基因蛋白表达及Caspase-3的活性。在CPB前、缺血90 min、再灌注30、60、120 min采静脉血检测血MDA、SOD水平。结果原位化学法测得心肌细胞凋亡率组2(10．46±0．91)%、组3(9．68±0．59)%和组4(11．35±1．37)%显著低于组1(19．75±1．81)%(P＜0．05)；流式细胞法测得Fas,Caspase-3荧光表达指数(FI),组2(1．24±0．13和1．32±0．13)、组3(1．27±0．07和1．33±0．08)和组4(1．27±0．14和1．31±0．12)显著低于组1(1．74±0．11和1．99±0．12)(P＜0．05)；与组1相比,MDA血浆浓度组2、组3和组4显著低于组1,而SOD浓度却显著高于组1(P＜0．05)。组2、组3和组4上述指标差异无统计学意义(P＞0．05)。结论Fas、Caspase-3表达改变参与了心肌细胞凋亡及缺血再灌注损伤过程；缺血后处理可以明显减少心肌细胞凋亡,抑制缺血再灌注损伤。心肌细胞凋亡的减少与Fas基因蛋白的下调、抑制Caspase-3活性及氧化应激有关；缺血后处理与缺血预处理相比可以同等程度的减少心肌细胞凋亡。本实验未观察到缺血预处理和缺血后处理的叠加作用。
Objective To investigate the effect of ischemic postconditioning on Fas gene protein expression and Caspase-3 activity in porcine cardiomyocytes. Methods Twenty-four small Yorkshire pigs weighing 35-40 kg were randomly divided into four groups. In group 1 (ACC group, n = 6), CPB was circulated for 45 min in parallel, blocking the aorta for 90 min and reopening the aorta for 120 min. Group 2 (pre-con group, n = 6) Cardiac ischemic preconditioning (blocking the ascending aorta for 5 min, opening 10 min, repeated 3 times) was performed before ascending aorta occlusion, and the rest of the treatment was the same as in group 1. Group 3 (post-con group, n = 6) After 45 min of reperfusion, the aorta was occluded for 90 min and then reperfused for 120 min after the aorta was opened. After reperfusion, ischemic postconditioning was performed (30 s after the ascending aorta was occluded and 3 times for 3 min ); Group 4 (pre-con + post-con group, n = 6): Cardiac ischemic preconditioning was performed before ascending aorta occlusion (ascending aorta blocked for 5 min, The aorta was transected for 90 min. The aorta was reperfused for 120 min. After reperfusion, myocardial ischemic postconditioning was performed (blocking the ascending aorta for 30 s, opening for 30 s and repeating 3 times for 3 min). At the end of reperfusion, take the right ventricular full-thickness myocardium and fixed. The apoptosis of cardiomyocytes in each group was observed by TUNEL, the expression of Fas gene and the activity of Caspase-3 were detected by flow cytometry. Pre-CPB, ischemia 90 min, reperfusion 30,60,120 min venous blood was collected to detect blood MDA, SOD levels. Results The rate of apoptotic cells in group Ⅱ was (10.46 ± 0.91)%, group 3 (9.68 ± 0.59)% and group 4 (11.35 ± 1.37)%, respectively (P <0.05). The expression of Fas, Caspase-3 and FI in group 2 were significantly lower than those in group 1 (19.75 ± 1.81)% 13 and 1.32 ± 0.13), group 3 (1.27 ± 0.07 and 1.33 ± 0.08) and group 4 (1.27 ± 0.14 and 1.31 ± 0.12) (1.74 ± 0.11 and 1.99 ± 0.12) in group 1 (P <0.05). Compared with group 1, the plasma concentrations of MDA in group 2, group 3 and group 4 were significantly lower than those in group 1 Group 1, while the SOD concentration was significantly higher than Group 1 (P <0.05). Group 2, Group 3 and Group 4 above indicators no significant difference (P> 0.05). Conclusion The changes of Fas and Caspase-3 are involved in the process of cardiomyocyte apoptosis and ischemia-reperfusion injury. Ischemic postconditioning can significantly reduce the apoptosis of myocardial cells and inhibit the injury of ischemia-reperfusion. The decrease of apoptosis of cardiomyocytes is related to downregulation of Fas gene protein, inhibition of Caspase-3 activity and oxidative stress. Compared with ischemic preconditioning, ischemic postconditioning can reduce cardiomyocyte apoptosis to the same extent. This experiment did not observe the superposition of ischemic preconditioning and postconditioning.