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目的建立造血干细胞(HSCs)体外衰老模型,探讨其衰老的相关生物学调控机制,为寻找延缓HSC衰老方法奠定基础。方法用免疫磁性分选法分离、纯化小鼠Sca-1+HSCs,用流式细胞术鉴定分选细胞纯度,免疫荧光检测分选细胞Sca-1+表达。用三丁基过氧化氢(t-BHP)100μmol/L诱导Sca-1+HSCs 6h,建立HSCs衰老体外模型。采用造血祖细胞集落培养,细胞周期分析和衰老相关β-半乳糖苷酶(SA-β-gal)染色观察衰老HSCs的生物学特点。DNA印迹法(Southern blotting)与TRAP-PCR SYBR Green染色法检测HSCs端粒长度和端粒酶活性。RT-PCR法检测衰老相关基因p16Ink4a、p19Arf、p53、p21Cip1/Waf1 mRNA的表达水平。结果免疫磁性分选法分离、纯化的Sca-1+HSCs纯度可达(87.33±1.25)%。100μmol/L的t-BHP作用Sca-1+HSCs 6h,其体外培养形成造血祖细胞集落能力,自我更新及多向分化潜能显著低于对照组,处于G1期细胞比例增加,SA-β-gal染色阳性细胞数增加。衰老Sca-1+HSCs的端粒缩短,端粒酶活性下降,p16Ink4a、p19Arf、p53、p21Cip1/Waf1mRNA的表达水平明显增高。结论用t-BHP能建立Sca-1+HSCs体外细胞衰老模型,提示p16Ink4a-Rb通路和p19Arf-Mdm2-p53-p21Cip1/Waf1通路在t-BHP诱导Sca-1+HSCs衰老过程中发挥重要作用。
OBJECTIVE: To establish an in vitro aging model of hematopoietic stem cells (HSCs) and to explore the related biological regulatory mechanisms of aging, so as to lay the foundation for finding a way to delay HSC aging. Methods Sca-1 + HSCs were isolated and purified by immunomagnetic sorting. The purity of sorted cells was identified by flow cytometry. The expression of Sca-1 + was detected by immunofluorescence. Sca-1 + HSCs were induced by tributyl hydroperoxide (t-BHP) 100μmol / L for 6h, and established HSCs aging model in vitro. The biological characteristics of aged HSCs were observed by colony culture of hematopoietic progenitor cells, cell cycle analysis and aging-related β-galactosidase (SA-β-gal) staining. The telomere length and telomerase activity of HSCs were detected by Southern blotting and TRAP-PCR SYBR Green staining. The expression of p16Ink4a, p19Arf, p53, p21Cip1 / Waf1 mRNA in senescence-related genes was detected by RT-PCR. Results The purity of Sca-1 + HSCs isolated and purified by immunomagnetic separation was (87.33 ± 1.25)%. Sca-1 + HSCs treated with 100μmol / L t-BHP for 6h showed the ability of hematopoietic progenitor cells to colonize in vitro, and the potential of self-renewal and multidirectional differentiation was significantly lower than that of the control group. The proportion of cells in G1 phase increased. Dyed positive cells increased. The telomere of aged Sca-1 + HSCs was shortened and the telomerase activity was decreased. The expression of p16Ink4a, p19Arf, p53, p21Cip1 / Waf1mRNA was significantly increased. Conclusion The cell aging model of Sca-1 + HSCs can be established by t-BHP, which indicates that p16Ink4a-Rb pathway and p19Arf-Mdm2-p53-p21Cip1 / Waf1 pathway play an important role in the senescence of Sca-1 + HSCs induced by t-BHP.