荧光内参在校正Caspase-3／7活性检测偏差中的应用

目的改进Caspase-3／7活性检测方法。方法在顺铂诱导的HeLa细胞凋亡模型中,分别利用传统的和改良的实验方法检测Caspase-3／7的活性变化,并与Western印迹实验结果进行方法学比较。结果改良的实验方法显示不同剂量顺铂诱导下,HeLa细胞Caspase-3／7活性有剂量依赖性增高,与Western印迹实验结果相一致,但传统实验方法显示HeLa细胞Caspase-3／7活性呈现先增高后降低的趋势。结论由于参测细胞数不同,导致这种Caspase-3／7活性检测方法不能真实反映细胞凋亡程度。本研究成功建立了一种新的改良型Caspase-3／7活性检测方式,这种检测方法可以排除不同凋亡诱导方式诱导细胞凋亡时所产生的因参测细胞数不同所造成的Caspase-3／7活性检测的误差。

Application of Internal Control Reniila Luciferase in Unbiased Estimation of Caspase-3/7 Activity Assay for Apoptosis

Objective To improve the accuracy of Caspase-3/7 activity assay. Methods In cisplatin induced apoptotic Hela cell model, Caspase-3/7 activity of Hela cells was determined by traditional method or modified method. Meanwhile, cleaved PARP was detected by Western blot to determine apoptosis induction. Results The results of modified assay showed that Caspase-3/7 activity was increased in a cisplatin dose dependent manner, which was consistent with the Western blot result. However, the results of traditional assay showed that Caspase-3/7 activity of Hela cells was firstly increased but then declined, displaying a bias due to decreased cell numbers in the high dose of eisplatin. Conclusion Because the cell numbers involved in the assay were different, the traditional Caspase-3/7 activity assay is not reliable to reflect the apoptotic extent of tested samples. Our newly established Caspase-3/7 activity assay with internal control may correct the deviations of Caspase-3/7 activity assay caused by difference in cell numbers among samples, thus providing more accurate and reliable results.