摘要
目的 建立一种简便、高效的无细胞凋亡体系 ,探讨外源细胞核在其中的凋亡过程及z VAD fmk对其的抑制作用。方法 超速离心制备非洲爪蟾卵无细胞体系 ,用细胞色素C激活为凋亡体系。应用荧光显微镜、琼脂糖凝胶电泳、ApoAlertCaspase 3ColorimetricAssay等方法检测外源细胞核在无细胞凋亡体系中的凋亡过程及z VAD fmk的抑制作用。结果 制备的爪蟾卵无细胞体系可由细胞色素C激活为凋亡体系 ,外源加入的细胞核呈现典型的形态学变化 ,总DNA特异降解形成DNAladder。体系内内源特异核酸酶被激活。z VAD fmk可抑制DNAladder的形成。凋亡特异蛋白酶XCPP32活性明显升高。结论 本实验建立的无细胞凋亡体系可以高效的诱导外源细胞核凋亡 ,重现体内过程。
Aim To find a convenient and highly effective cell-free apoptotic system and investigate the apoptotic process of mouse liver nuclei in the system and the inhibitory effects of z-VAD-fmk on apoptosis. Methods The cell-free system was prepared by ultracentrifugation of Xenopus eggs and activated by cytochrome C. Fluorescent microscope, argrose gel electrophoresis and apoalert Caspase-3 colorimetric assay were applied to detect the apoptotic process of isolated nuclei and the inhibitory effecs of z-VAD-fmk on the system. Results The prepared cell-free system was shown to be activated by cytochrome C. The exogenous mouse liver nuclei presented typical morphologic changes and DNA ladder formation was observed which could be inhibited by z-VAD-fmk. Specific endonuclease was activated and XCPP32 activity was significantly increased in the system. Conclusion The prepared cell-free apoptotic system can effectively induce isolated nuclei apoptosis and mimick the process in vivo .
出处
《药学学报》
CAS
CSCD
北大核心
2003年第1期10-14,共5页
Acta Pharmaceutica Sinica
