人参皂苷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

Relationship between ginsenoside Rg1-induced senescence of human erythroleukemia K562 cells and p16-Rb signal pathway

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 ginsenoside Rgl-induced senescence of human erythroleukemia K562 cells andpl6-Rb signal pathway. Methods K562 cells were treated with Rgl at various concentrations（0, 5, 10, 20, 40 and 80 μmol/L） for various time （24, 48 and 72 h）, based on which the optimal concentration and time were screened by MTY method. K562 cells were treated with Rgl at optimal concentration for various hours, and determined for cell cycle by flow cytometry. K562 cells were treated with Rgl at optimal concentration for optimal hours, and determined for colony formation ability by colony culture method. The percentage of positive cells was determined by SA-β-Gal staining. The uhrastructure of cells was observed by transmis- sion electron microscopy, while the telomere length was determined by Southern blot, and the expressions of senescence-related pro- teins P16 and RB by Western blot. Results Rgl significantly inhibited the proliferation of K562 cells in vitro, of which the optimal working concentration and time were 20 μmol/L and 48 h respectively. Compared with those in routine culture as control group, the cell cycle of K562 cells after treatment with 20 μmol/L Rgl for 48 h was arrested in G2/M phase, while the colony formation ability decreased significantly （P 〈 0. 05）, the percentage of cells positive in SA-β-Gal staining increased significantly （P 〈 0. 05）. The ob- servation of ultrastructure showed phenotypes of senescence phenotypes, such as increased sizes of cells, increased lysosomes and mi- tochondrial in size and number, quick shortening of telomere length （P 〈 0. 05） and up-regulation of P16 and RB expressions （P 〈 0. 05）. Conclusion Rgl induced the senescence of K562 cells, which might be associated with the activation ofpl6-Rb signal pathway.