比色法检测Caspase-3相对活性

Colorimetric assay of Caspase-3 relative activity

目的建立比色法检测Caspase-3相对活性的方法。方法以H9c2(2-1)细胞缺氧/复氧损伤为凋亡诱导模型,采用比色法检测Caspase-3相对活性,并使用吖啶橙/溴化乙啶(AO/EB)染色法进行平行的方法学比较。结果H9c2(2-1)细胞缺氧/复氧组与对照组相比Caspase-3相对活性升高了0.48倍,差异具有统计学意义(1.48±0.16 vs 1.00±0.06,P<0.01);缺氧/复氧组细胞经AO/EB染色出现凋亡的形态学特征。结论本研究成功建立了Caspase-3相对活性的比色检测方法,与试剂盒方法相比大大降低了检测成本,同时也为检测Caspases家族其他蛋白酶的活性提供了方法学依据。

Objective To establish the colorimetric assay of Caspase-3 relative activity. Methods H9c2 （2-1） cell apoptosis was induced by hypoxia-reoxygenation injury. Caspase-3 relative activity in these cells was detected using colorimetric assay. Meanwhile, apoptosis of H9c2 （2-1） cells was observed using acridine orange/ethidium bromide （AO/EB） staining as parallel comparison of methodology. Results The Caspase-3 relative activity in hypoxia-reoxy- genation （H/R） group was significantly increased by 48% compared with control group （1.48±0.16 vs 1.00±0.06, P〈0.01）. AO/EB staining also revealed changes in morphology typical of apoptosis in H/R-injured H9c2 （2-1） cells. Conclusion Colorimetric assay of Caspase-3 relative activity was successfully established, which may be less expensive than the use of Caspase-3 activity assay kits, and may provide a basis for measuring relative activity of other Caspases.