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目的 探讨眼镜蛇毒细胞毒素CTX-d诱导NB细胞凋亡的机制。方法 MTT法测定CTX-d体外细胞毒作用,电镜、流式细胞仪观察CTX-d对NB4细胞的诱导凋亡作用,流式细胞仪检测NB4细胞线粒体膜电位的变化,Western-blotting测定胞浆细胞色素C及caspase-9、caspase-3的变化。结果 CTX-d作用NB4细胞6、12h的IC50分别为1.8、1.35μg/mL;CTX-d引起NB4细胞线粒体肿胀、核固缩等形态学改变;诱导NB4细胞出现亚G1期的凋亡峰,且有时效及量效关系;CTX-d(1.0μg/mL)作用0.5h,NB4细胞线粒体膜电位已开始下降,同时在胞浆中检测到细胞色素C,显示细胞色素C已由线粒体释放入胞浆;caspase-9酶原的量在CTX-d(1.0μg/mL)作用1h时开始下降,而活化的caspase-3片断在0.5h即检测到,表明除了通过caspase-9,CTX-d还可能通过其他途径激活caspase-3。结论 CTX-d可通过降低线粒体膜电位、细胞色素C释放,激活caspase-9和caspase-3,从而诱导NB4细胞凋亡,还可能通过其他途径激活caspase-3,参与凋亡作用。
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[Abstract]
Objective To investigate the effects of CTX—d from venoms of cobra(Naja naja atra)on inducing NB4 apoptosis and its mechanism.Methods MTT was used to detect the antitumor effect of CTX—d in vitro;Electron microscope and flow cytometry were used to observe the apoptotic inducing effect Df CTX—d in NB4 cells;Mitochondrial transmembrane potential change(△甲m)was analyzed by flow cytometry;The levels of caspase一9,caspase一3,and cytochrome C in the cytosol fraction were analyzed by Western blotting.Results The IC50 values of CTX—d affected on NB4 eell for 6 and 12 h were 1.8 and 1.35}£g/mL,respectively.CTX—d could induce morphological changes,such as condensed chromatin and swelling mitochondria in NB4 cells.Analyzed by flow cytometry,CTX-d induced apoptosis in NB4 cells evidenced by increasing sub G1 cell population in a dose-and time—dependent-manner.The mitochondrial membrane potential of NB4 cells had already decreased when incubated with CTX—d(1.0肛g/mL)for 0.5 h,and cytochrome C in the cytosol was detected simultaneously,which indicated the release of cytochrome C from mitochondria to cytos01.The caspase-9 was activated initially at 1 h after 1.0弘g/mL CTX—d treatment,whereas the cleavage of caspase一3 was detected at 0.5 h.This suggested that some other mechanism may be involved in caspase一3 activation.Conclusion The results suggest that the JOSS of mitochondrial membrane potential and the release of cytochrome C from the mitochondria into the cytosol are the early events of CTX—d on NB4 apoptosis.Once release into the cytos01.cytochrome C precedes the activation of caspase—·9 and·—3 to leading to the apoptosis and there are maybe some other mechanism involved in caspase-3 activation.
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