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目的探讨人参皂苷Rg1(以下简称Rg1)诱导人红白血病K562细胞株衰老与p16-Rb信号通路的相关性。方法以不同浓度的Rg1(0、5、10、20、40和80μmol/L)作用于K562细胞不同时间(24、48、72 h),MTT法筛选Rg1抑制K562细胞增殖的最佳作用浓度及作用时间,以该浓度干预K562细胞不同时间,流式细胞术检测细胞的细胞周期;以最佳浓度Rg1干预K562细胞最佳时间,集落培养法检测细胞集落形成能力;衰老相关-β-半乳糖苷酶(SA-β-Gal)染色检测阳性细胞百分率;透射电镜观察细胞的超微结构;Southern blot检测细胞的端粒长度;Western blot检测细胞中P16和RB蛋白的表达。结果 Rg1在体外可明显抑制K562细胞增殖,其最佳作用浓度及作用时间分别为20μmol/L和48 h;经20μmol/L Rg1诱导48 h的K562细胞与常规培养对照组相比,细胞出现G2/M期阻滞(P<0.05),集落形成能力明显减弱(P<0.05),SA-β-Gal染色阳性率增加(P<0.01),细胞体积增大,且溶酶体,线粒体体积增大,数目增多,端粒缩短加速(P<0.05),P16和RB蛋白表达上调(P<0.05)。结论 Rg1能诱导人红白血病K562细胞株衰老,可能与Rg1激活了p16-Rb信号通路有关。
Objective To investigate the relationship between the aging of human erythroleukemia K562 cell line and p16-Rb signaling pathway by ginsenoside Rg1 (hereinafter referred to as Rg1). Methods Different concentrations of Rg1 (0, 5, 10, 20, 40 and 80 μmol / L) were applied to K562 cells at various times (24, 48 and 72 h). MTT assay was used to screen the optimal concentration of Rg1 to inhibit the proliferation of K562 cells. The effect of Rg1 on the proliferation of K562 cells was observed by flow cytometry. The best time of K562 cells intervention by Rg1 was detected by flow cytometry. The colony-forming ability of colony-forming assay The percentage of positive cells was detected by SA-β-Gal staining. The ultrastructure of the cells was observed by transmission electron microscopy. The telomere length was detected by Southern blot. The expression of P16 and RB protein was detected by Western blot. Results Rg1 could inhibit the proliferation of K562 cells in vitro. The optimal concentration and duration of Rg1 were 20 μmol / L and 48 h, respectively. Compared with the normal control group, K562 cells induced by 20 μmol / L Rg1 exhibited a G2 (P <0.05). The positive rate of SA-β-Gal staining increased (P <0.01), the cell volume increased, and the volume of lysosome and mitochondria increased (P <0.05), and P16 and RB proteins were up-regulated (P <0.05). Conclusion Rg1 can induce the senescence of human erythroleukemia K562 cell line, which may be related to the activation of p16-Rb signaling pathway by Rg1.