摘要目的 探讨人参皂苷Rg1调控造血干细胞(HSC)衰老的机制,为寻找延缓HSC衰老方法 提供理论指导和实验依据.方法 免疫磁性分选法分离纯化小鼠Sca-1+HSC后分5组:对照组、衰老组、Rg1组、Rg1治疗衰老组、Rg1延缓衰老组.β-半乳糖苷酶(SA-β-Gal)染色、细胞周期测定和造血祖细胞混合集落(CFU-Mix)培养观察Rg1延缓Sca-1+HSC衰老的生物学作用.反转录-聚合酶链反应(RT-PCR)检测衰老相关基因p161NK4a、p19Arf、p53、p21Cipl/Wafl mRNA的表达.Western印迹检测p16INK4a、p21 Cipl/Wafl、细胞周期蛋白(cyclin)D1、cyclin E、细胞周期蛋白依赖性激酶(CDK)4、CDK2蛋白表达.结果 Rg1治疗衰老组及Rg1延缓衰老组Sca-1+HSC的SA-β-Gal染色阳性率(30.1%±2.4%,21.5%±2.8%)低于衰老组(69.5%±5.0%);G1期细胞比例(81.4%±1.2%,78.2%±1.4%)低于衰老组(87.5%±4.0%);生成CFU-Mix数[(8.0±2.2)个/104Sca-1+HSC、(9.2±1.8)个/104 Sca-1+HSC]高于衰老组[(3.0±1.6)个/104Sca-1+HSC];Rg1治疗衰老组及Rg1延缓衰老组Sca-1+HSC的p16INK4a、p19Arf、p53、p21 Cipl/Wafl mRNA及p16INKa、p21 Cipl/Wafl、cyclinD1蛋白表达均下调(均P＜0.01),CDK4、CDK2、cyclinE蛋白表达均上调(均P＜0.01).Rg1延缓衰老组各检测指标均较Rg1治疗组变化明显.结论 Rg1具有延缓及治疗Sca-1+HSC衰老的作用,p16INK4a-Rb及p19Aarf-Mdm2-p53-p21Cipl/Wafl信号通路在其中可能发挥重要作用.

abstractsObjective To investigate the underlying mechanism of ginsenoside Rg1 in the regulation of hematopoietic stem cell (HSC) senescence so as to provide the theoretic and experimental foundations for searching the methods of how to delay its senescence. Methods Sca-1 + HSC was isolated by magnetic cell sorting (MACS) and divided into control, aged, Rg1, Rg1 treatment aged and Rg1 delayed aged groups. The cellular changes were observed by senescence-associated β-galactosidase ( SA-β-Gal ) staining. And cell cycle assay and culture of mixed hematopoietic progenitor cell were used to investigate the effect of ginsenoside Rg1 to delay Sca-1 + HSC senescence. The expressions of p16INK4a, p19Arf, p53 and p21Cipl/Wafl mRNA were detected by reverse transcription-polymerase chain reaction ( RT-PCR ). The expressions of p16INK4a, p21Cipl/Wafl , cyclinD1, cyclinE, CDK2 and CDK4 protein were examined by Western blot. Results In the Rg1 treatment and delayed aged groups, the percentage of positive SA-β-Gal-expressing cells was lower than that of the aged group [30. 1% ± 2. 4%, 21.5% ± 2. 8% vs 69. 5% ± 5. 0%]; the number of cells in G1 phase was lower than that of the aged group [81.4% ± 1. 2%, 78.2% ± 1. 4% vs 87. 5% ±4. 0%]; but the number of colony for the mixed hematopoietic progenitor was higher than that of the aged group [(8. 0 ±2. 2)/104 Sca-1 + HSC, (9. 2 ± 1. 8)/104 Sca-1 + HSC vs (3.0 ± 1. 6)/104 Sca-1 +HSC].As compared with the aged group, the expressions of p16INK4a, p19Arf, p53, p21cipl/Wafl mRNA and p16INK4a,p21Cipl/Wafl, cyclinD1 protein were down-regulated (all P ＜ 0. 01 ) while the expressions of CDK4, CDK2and cyclinE protein up-regulated in Rg1 treatment aged and Rg1 delayed aged groups ( all P ＜0. 01 ). The changes of the Rg1 delayed aged group were significantly marked than those of the Rg1 treatment aged group. Conclusions Rg1 can effectively delay the t-BHP-induced senescence of HSCs. Both p16INK4a-Rb and p19Arf-p53-p21Crp/Wafl may play an important role in the signaling pathway.