非选择性冠状动脉微栓塞对大鼠心肌细胞凋亡及左室功能的影响

Effects of non-selectivity coronary microembolization on myocardial apoptosis and the left ventricular function of rats

目的:探讨非选择性冠状动脉微栓塞(CME)对大鼠心肌细胞凋亡及左室功能的影响。方法:采用雄性SD大鼠56只,体重280～320g。随机分为假手术组(Sham组,24只)和CME组(32只)。CME组夹闭升主动脉同时自左室腔注入自体微血栓混悬液,Sham组则注入等容量的0.9%氯化钠溶液。术后3d、7d、14d及28d(每个时间点CME组8只,Sham组6只)采用苏木精-伊红染色观察心肌组织病理学改变,RealTimePCR(实时定量聚合酶链式反应)检测心肌细胞Bcl-2、Bax、Fas、Fas-L等凋亡调控基因mRNA的表达,采用Western blot蛋白印迹法检测Caspase3、Caspase9和裂解PARP蛋白的表达,TUNEL(脱氧核糖核苷酸末端转移酶介导的缺口末端标记法)测定凋亡细胞,应用有创血流动力学检测大鼠左心功能的变化。结果:①苏木精-伊红染色显示,CME术后可见心内膜下多中心小灶性心肌缺血、坏死伴白细胞浸润。②术后7d,与Sham组相比较,CME组Bax、Fas及Fas-LmRNA的表达水平明显增高,Bcl-2/Bax的比值明显降低(0.18±0.04:2.98±0.49P<0.01);Caspase-3、Caspase-9及裂解PARP蛋白表达明显升高(0.762±0.129:0.133±0.027,0.295±0.023:0.067±0.020,0.992±0.146:0.386±0.074,均P<0.01)。③术后7dCME组凋亡指数较Sham组明显增高(17.2±1.9:1.2±0.6,P<0.01)。④LVSP及±dp/dtmax较Sham组明显降低(P<0.01)。结论:CME早期即可通过改变凋亡调控基因的平衡,启动、激活Caspase级联反应,促进心肌细胞凋亡,降低左室功能。

Objective：To study The effects of non-selectivity coronary microembolization （CME） on myocardial apoptosis and left ventricle function of rats.Method：A total of 56 male Sprague Dawley rats were randomized into control group （n=32） and sham-operated group （n=24）.The control group was underwent injection of homologous microthrombotic particle suspension into left ventricle when clamping the ascending aorta and the sham-operated group was injected with saline.The rats were sacrificed at day 3,7,14 and 28 after operation.The pathology was observed by HE dyeing.The mRNA of Bcl-2,Bax,Fas,Fas-L and GAPDH which was used as the intracomparison,were evaluated by Real Time PCR.The ratios of Bcl-2/Bax also be compared.The protein expression of Caspase-3,Caspase-9 and the cleaved PARP were detected by western blot.Myocardial apoptosis and left ventricular function were checked by TUNEL staining and hemodynamics.Result：In the control group,the histological examination showed that there were multi-focal myocardial ischemia,necrosis with inflammatory cells infiltration in subendocardial region.Compared with the sham-operated group,the mRNA of Bcl-2,Bax,Fas and Fas-L in-creased whereas the ratio of Bcl-2/Bax markedly decreased in control group （day 7：0.18±0.04 vs 2.98±0.49,P0.01）.Simultaneously,the protein expression of Caspase-3,Caspase-9 and the cleaved PARP enhanced.The myocardial apoptosis index elevated（day 7：17.2±1.9 vs 1.2±0.6,P0.01） and the left ventricular function decreased significantly （P0.01）.The fastigium of all the changes was occurred at day 7.Conclusion：The CME could promote myocardial apoptosis and depress the left ventricular function by altering the balance of the apoptosis gene and activating the caspase system at early period.