TNFα单克隆抗体对(狗)体外循环术后心肌细胞凋亡的影响

摘要

目的观察抗TNF-α单克隆体(MaTA)对(狗)常规CPB 术后心肌细胞凋亡及其对caspase-3酶活性的影响.方法 10条用于模拟常规CPB手术的成年家犬随机分为对照组和MaTA处理组,每组各5条.MaTA处理组动物CPB前和开放升主动脉后分别经静脉注射MaTA(0.5μg/100ml) 对照组动物不用MaTA处理.留取CPB前、中及CPB后3h的全层左心室标本,液氮保存,用原位TUNEL技术检测心肌细胞凋亡并记录每105个心肌细胞中凋亡阳性细胞数目.用Z-DEVD-AMC 底物降解方法测定caspase-3 酶活性.结果 CPB前和CPB中两组的心肌细胞凋亡数及caspase-3酶活性在组内和组间均无显著性差异(P＞0.05).CPB术后3h两组的心肌细胞凋亡数均较CPB前和CPB中明显增高(P分别＜0.05),而处理组CPB术后心肌细胞凋亡数明显低于对照组(P＜0.05);CPB 术后3h两组的caspase-3酶活性也均较术前和术中显著性增高(P＜0.01,0.01,0.05和0.05),与对照组比较,MaTA处理组caspase-3活性也明显受到抑制(P＜0.02).结论抗TNFα单克隆抗体可以明显地降低CPB术后心肌细胞凋亡.这一抑制作用伴随有对caspase-3酶活性的抑制.%Objective　To investigate the effects of monoclonal anti-TNFα antibody on cardiomyocyte apoptosis and activity of caspase-3 enzyme in dog hearts following standard cardiopulmonary bypass(CPB).Methods　Ten healthy adult dogs of either sex mimicking clinical standard CPB process were randomized into control group(n=5) and monoclonal anti-TNFα antibody treated group(n=5).Dogs in the latter group received a bolus(0.5 μg/kg,iv) of monoclonal anti-TNFα antibody 10 min before the clamp of the ascending aorta and just after the reperfusion of the heart, and 0.1 μg/100ml of monoclonal anti-TNFα antibody was also added into the cold cardioplegia for dogs in this group. Samples of the left ventricle(LV) were obtained before ,during and 3 hrs after CPB and stored in liquid nitrogen until assay. In situ TUNEL technique was used to detect the apoptotic cells and the number of apoptotic positive cells in per 105 myocytes was counted.The activity of caspase-3 enzyme was measured by cleavage of Z-DEVD-AMC substrate.Results　Both the number of apoptotic myocytes and the activity of caspase-3 enzyme were not significantly different between before and during CPB and between groups (P＞0.05).The number of apoptotic myocytes at 3 hrs after CPB in both control and monoclonal anti-TNFα antibody treated groups was significantly increased compared with that before and during CPB (P<0.01,respectively), while it was significantly reduced in the monoclonal anti-TNF α antibody treated with that of control group (P＜0.05).The activity of caspase-3 enzyme at 3 hrs after CPB in both control and monoclonal anti-TNFα antibody treated with that before and during CPB (P<0.01,0.01,0.05 and 0.05,respectively) and was significantly inhibited by monoclonal anti-TNFα antibody treatment (vs control group P<0.01). Conclusion　Treatment with monoclonal anti-TNFα antibody significantly reduced cardiomyocyte apoptosis in dog heats undergoing standard CPB. This study also demonstrates that the effect of the reduction of the myocyte apoptosis is associated with the inhibition of activity of caspase-3 enzyme.