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目的:研究真菌Polyporus sp.M05多糖对白血病细胞株HL-60增殖抑制及诱导凋亡作用,并探讨其分子机制。方法:活细胞计数法检测多糖的增殖抑制作用;倒置显微镜下直接观察细胞形态;PI染色流式细胞术检测多糖诱导周期阻滞及凋亡作用;RT-PCR检测凋亡相关基因。结果:多糖对HL-60细胞有增殖抑制作用,并随着浓度增大,作用增强;显微镜下观察细胞有明显的凋亡小体。流式细胞术检测细胞有明显G0/G1期阻滞及凋亡峰的出现,并随着时间越长和浓度的增大凋亡阳性率比例越高,与阴性对照相比差异有统计学意义(P<0.01);RT-PCR检测到Bcl-2基因下调,Bax、Fas及Caspase-9基因上调。结论:Polyporus sp.M05中提取的多糖对白血病细胞株HL-60有增殖抑制和诱导凋亡作用,分子机制与Bcl-2基因下调,Bax、Fas及Caspase-9基因上调有关,线粒体途径及死亡受体途径均参与了此凋亡过程。
OBJECTIVE: To study the effects of polysaccharide Polyporus sp. M05 on the proliferation and apoptosis of leukemia cell line HL-60 and to explore its molecular mechanism. Methods: The proliferation inhibition of polysaccharides was detected by viable cell counting method. The morphological changes of cells were observed under inverted microscope. The cell cycle arrest and apoptosis were detected by flow cytometry. The apoptosis related genes were detected by RT-PCR. Results: Polysaccharide inhibited the proliferation of HL-60 cells, and with the increase of concentration, the effect was enhanced. The apoptotic bodies were observed under microscope. Flow cytometry showed significant G0 / G1 phase arrest and peak of apoptosis, and with the increase of time, the positive rate of apoptosis was higher, which was significantly different from the negative control (P <0.01). The down-regulation of Bcl-2 gene and the up-regulation of Bax, Fas and Caspase-9 genes were detected by RT-PCR. CONCLUSION: Polysaccharide extracted from Polyporus sp. M05 can inhibit proliferation and induce apoptosis of leukemia cell line HL-60. The molecular mechanism is related to down-regulation of Bcl-2 gene, up-regulation of Bax, Fas and Caspase-9 gene, mitochondrial pathway and death Receptor pathways are involved in this process of apoptosis.