目的 研究三七总皂苷(PNS)对K562细胞增殖、凋亡、周期的影响及其相关分子机制.方法 采用MTT法检测PNS对K562细胞增殖的影响;AO/EB双荧光染色法及Annexin V-FITC/PI双染色法观察细胞凋亡及死亡状况;流式细胞术检测K562细胞的周期变化;RT-PCR检测mTOR信号通路主要分子mRNA的表达变化;Western blot检测cleaved caspase-3蛋白、cyclin D1蛋白及mTOR信号通路主要蛋白及磷酸化蛋白的表达量变化.结果 100~800μg/mL的PNS作用于K562细胞后能够抑制细胞增殖.PNS能够上调cleaved caspase-3蛋白表达,促使K562细胞发生凋亡.并且下调cyclin D1蛋白表达,促使K562细胞阻滞在G0/G1期.同时,PNS能够抑制K562细胞mTOR mRNA表达,降低mTOR蛋白和磷酸化蛋白p-mTOR(Ser2448)、p-p70S6K(Thr229/389)及p-4E-BP1(Thr37/46)的表达,从而抑制mTOR信号通路活性.结论 PNS对体外培养K562细胞有抑制增殖,促进凋亡,使其发生周期阻滞的作用.其机制可能与PNS抑制mTOR信号通路活性、上调cleaved caspase-3蛋白表达及抑制cyclin D1蛋白表达有关.%Objective To investigate the effects of Panax notoginseng saponins (PNS) on the proliferation, apoptosis and cell cycle of K562 cells and explore the molecular mechanisms underlying these effects. Methods PNS-induced growth inhibition of K562 cells was detected by MTT assay;the cell apoptosis was evaluated by AO/EB staining and Annexin V-FITC/PI staining;flow cytometry was used to detect cell cycle changes in the treated cells. The mRNA expressions of the molecules in mTOR signaling pathway were examined by RT-PCR, and the cellular expressions of cleaved caspeas-3, cyclin D1 and major proteins in mTOR signaling pathway were detected using Western blotting. Results MTT assay showed that treatment with 100-800μg/mL PNS significantly inhibited the proliferation, promoted the cell apoptosis, and caused cell cycle arrest in G0/G1 phase in K562 cells. Western blotting revealed increased protein expression of cleaved caspase-3 and decreased expression of cyclin D1 in PNS-treated cells, in which the proteins expressions of mTOR, p-mTOR, p-p70S6K and p-4E-BP 1 and the mRNA expression of mTOR were all decreased. Conclusion PNS can inhibit the proliferation, induce apoptosis and cause cell cycle arrest in K562 cells possibly by up-regulating cleaved caspase 3 and down-regulating cyclin D1 and mTOR signaling pathway.
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