摘要
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 the aging model of hematopoietic stem cells（HSCs） and investigate its related biological mechanism. The purpose is to build the foundation for searching the methods of delaying HSCs aging. Methods Sca-1 ＋ HSCs were isolated and purified by magnetic activated cell sorting （MACS）. The purity of separated cells was analysed by flow cytometry （FCM） and the expression of Sca-1 ^＋ antigen was detected by immunofluorescence. Sca-1 ^＋ HSCs were induced by tert-butylhydroperoxide（ t-BHP, final concentration of 100 μmol/L） for 6 hours to establish the HSCs aging model in vitro. Biological characteristics of aging HSCs was evaluated by mixed hematopoietic progenitor cell culture, cell cycle assay and sensscence-associated β-galactosidase （SA-β-gal） cytocbemical staining. Telomere length and telomerase activity were detected by Southern blotting and TRAP-PCR-SYBR Green staining. By using reverse transcription polymerase chain reaction（RT-PCR） , the expression of p16^lnk4, p19^Arf, p53, p21^Cipl/Wafl mRNA was detected. Results The purity of separated Sca-1 ^＋ HSCs was （87.33 ± 1.25） %. After being cultured with 100 μmol/L t-BHP for six hours, the ability of aging Sca-1 ＋ HSCs to forming mixed hematopoietic progenitor colony, self-renewal and muhi-differentiation was decreased significantly. The number of aging Sca-1^ ＋ HSCs entered G1 phase of the cell cycle, the percentage of SA-β- gal positive cells and the expression of p16^Ink4a, p19^Arf, p53, p21^Cipl/WaflmRNA were increased. The telomere length was shorten and the telomerase activity was decreased. Conclusion The t-BHP can induce Sca-1 ^＋ HSCs senescence in vitro.The signal transduetion pathway of pl6I＂k4＂-Rb and p19A^f-Mdm2-p53-p21Cipl/Wafl might play a key role in the Sea-1 ^＋ HSCs senescence induced by t-BHP.
出处
《解剖学报》
CAS
CSCD
北大核心
2010年第5期686-692,共7页
Acta Anatomica Sinica
基金
国家自然科学基金资助项目(30973818)
重庆市科委自然科学基金重点项目(CSTC,2009BA5038)
