CAJ | 학술논문

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.
목적 연구삼칠총조감(PNS)대K562세포증식、조망、주기적영향급기상관분자궤제.방법 채용MTT법검측PNS대K562세포증식적영향;AO/EB쌍형광염색법급Annexin V-FITC/PI쌍염색법관찰세포조망급사망상황;류식세포술검측K562세포적주기변화;RT-PCR검측mTOR신호통로주요분자mRNA적표체변화;Western blot검측cleaved caspase-3단백、cyclin D1단백급mTOR신호통로주요단백급린산화단백적표체량변화.결과 100~800μg/mL적PNS작용우K562세포후능구억제세포증식.PNS능구상조cleaved caspase-3단백표체,촉사K562세포발생조망.병차하조cyclin D1단백표체,촉사K562세포조체재G0/G1기.동시,PNS능구억제K562세포mTOR mRNA표체,강저mTOR단백화린산화단백p-mTOR(Ser2448)、p-p70S6K(Thr229/389)급p-4E-BP1(Thr37/46)적표체,종이억제mTOR신호통로활성.결론 PNS대체외배양K562세포유억제증식,촉진조망,사기발생주기조체적작용.기궤제가능여PNS억제mTOR신호통로활성、상조cleaved caspase-3단백표체급억제cyclin D1단백표체유관.
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.