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研究了羊栖菜多糖(SargassumFusiformePolysaccharides,SFPS)诱导人大肠癌lovo细胞凋亡及凋亡过程中caspase-3、caspase-8、caspase-9的活性变化。MTT法检测SFPS对lovo细胞增殖的抑制率;通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡;应用Western印迹法测定caspase-3酶原和caspase-9的变化;RT-PCR检测caspase-3mRNA表达;caspase-3、caspase-8、caspase-9活性检测试剂盒观察caspase-3、caspase-8、caspase-9的活性改变。结果显示,SFPS对lovo细胞增殖有显著抑制作用,经形态变化、DNA条带和流式细胞分析,可见明显的细胞凋亡特征。SFPS处理lovo细胞后,发现caspase-3酶原蛋白表达降低,caspase-3mRNA高表达,并具有剂量和时间的依赖性。而在检测蛋白中,也发现caspase-9被激活进而形成具有活性的片段。另外,caspase的活性检测也进一步发现caspase-3、caspase-9的活性逐步增高。实验结果提示SFPS在体外诱导lovo细胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一,并且是通过激活启动caspase-9,进而激活下游效应caspase-3的级联反应来实现的。
The effects of Sargassum Fusiforme Polysaccharides (SFPS) on the apoptosis of human colorectal cancer LoVo cells and the changes of caspase-3, caspase-8 and caspase-9 during apoptosis were studied. The inhibitory rate of SFPS on the proliferation of lovo cells was detected by MTT assay. The apoptosis of lovo cells was detected by electron microscopy, agarose gel electrophoresis and flow cytometry. The changes of caspase-3 and caspase-9 were detected by Western blotting. The activity of caspase-3, caspase-8 and caspase-9 were detected by caspase-3, caspase-8 and caspase-9 activity detection kit. The results showed that SFPS significantly inhibited the proliferation of lovo cells. The morphological changes, DNA bands and flow cytometry showed obvious apoptosis characteristics. SFPS treatment lovo cells, found that caspase-3 reduced proenzyme protein expression, caspase-3mRNA expression, with a dose and time-dependent. In the test protein, caspase-9 is also found to be activated to form an active fragment. In addition, caspase activity test also further found caspase-3, caspase-9 activity gradually increased. Experimental results suggest that SFPS induces apoptosis of lovo cells in vitro, which may be one of the mechanisms by which SFPS inhibits tumor proliferation and is activated by activating caspase-9, thereby activating the cascade of downstream effects of caspase-3.