摘要
目的探讨Caspase-3特异性抑制剂在缺氧诱导的培养乳鼠心肌细胞凋亡中的作用及其相关基因的表达。方法将SD乳鼠的培养心肌细胞随机分为对照组、缺氧72 h组及Caspase-3抑制剂组(缺氧72 h前加入0.1μmol/L的Z-DEVD-fmk)3组。应用Western blot分析法和RT-PCR技术检测Caspase-3、PARP蛋白及Caspase-3mRNA的表达,同时利用流式细胞仪分析心肌细胞凋亡的变化;借助Caspase-3荧光分析试剂盒,荧光比色法检测心肌细胞凋亡过程中Caspase-3活性的变化。结果Caspase-3抑制剂组细胞凋亡百分率为(14.93±2.47)%,显著低于缺氧72 h组的(48.43±4.18)%,差异有高度统计学意义(P<0.01)。Caspase-3抑制剂组PARP及Caspase-3蛋白表达显著低于缺氧72 h组,差异有高度统计学意义(P<0.01)。RT-PCR分析显示,Caspase-3抑制剂组Cas-pase-3 mRNA的表达量较缺氧72 h组降低了62.05%,差异有高度统计学意义,Caspase-3活性检测显示,Caspase-3抑制剂组的Caspase-3活性较缺氧72 h组降低了52.85%,差异有高度统计学意义(均P<0.01)。结论Caspase-3抑制剂能明显抑制缺氧诱导的心肌细胞凋亡、Caspase-3蛋白和mRNA的表达、Caspase-3蛋白酶活性和PARP的裂解。
Objective To investigate the significance of Caspase-3 inhibitor in neonatal cardiomyocytes apoptosis induced by hypoxia. Methods The cultured Cardiomyocytes from 1-2 d newborn SD rats were randomly divided into 3 groups： control groups, ischaemic 72 h group and Caspase-3 inhibitor group [addition of Z-DEVD-fmk（0.1μmol/L） 72 h before ischaemic treatment]. Caspase-3, PARP mRNA and protein were detected by RT-PCR and Western blot analysis, the Caspase-3 activity was detected by caspase-3 fluorescent assay kit. Cardiomyocytes apoptosis was detected by flow cytometry. Results The flow cytometry analysis showed that the percentage of apoptosis cells in Caspase-3 inhibitor group was （ 14.93 ±2.47） %, and compared with the 72 h ischaemic group （48.43±4.18） % was significantly lower（P〈0.01）. RT-PCR and western blot analysis showed that Caspase -3mRNA, PARP and Caspase-3 protein expression in Caspase-3 inhibitor group were significantly lower than those in the 72 h ischeamic group （P 〈 0.01）. Caspase-3 activity analysis showed that caspase-3 activity in caspase-3 inhibitor group reduced by 52.85 % relative to the 72 h ischaemic group. Conclusions Caspase-3 inhibitor can efficiently inhibit apoptosis of neonatal cardiomyocytes after hy- poxia insult. Caspase-3 inhibitor can efficiently inhibit expression of Caspase-3 protein and mRNA in cultured neonatal cardiomyocytes after hypoxia insult. Caspase-3 inhibitor can efficiently inhibit activity of Caspase-3 in cultured neonatal cardiomyocytes after hypoxia insult.
出处
《苏州大学学报（医学版）》
CAS
北大核心
2007年第4期510-513,554,共5页
Suzhou University Journal of Medical Science
基金
卫生部招标课题资助项目(200308)
