Laboratory of Stem Cell and Tissue Engineering, Department of Histology and Embryology, Chongqing Medical University, Chongqin, China.
In Vitro Cell Dev Biol Anim. 2011 Feb;47(2):149-56. doi: 10.1007/s11626-010-9372-5. Epub 2010 Dec 4.
The objective of this study is to establish the aging model of murine hematopoietic stem cell (HSC) ex vitro and investigate its relative biological mechanism, aimed to build the foundation for searching the methods to delaying HSC aging. Sca-1(+)HSC were isolated and purified from murine bone marrow mono-nucleated cell by magnetic-activated cell sorting. The purity of separated cells was analyzed by flow cytometry (FCM) and the expression of Sca-1 was detected by immunofluorescence. Sca-1(+)HSC induced aging by tert-butylhydroperoxide (t-BHP, final concentration of 100 μmol/L) for 6 h to establish the murine HSC aging model in vitro. Biological characteristics of aging HSC were evaluated by mixed hematopoietic progenitor cell culture in vitro, cell cycle assay and senescence-associated β-galactosidase (SA-β-gal) cytochemical staining. Telomere length and telomerase activity were detected by southern blotting and telomere repeat amplification protocol-polymerase chain reaction (TRAP-PCR) augmentation. The expressions of p16(INK4a), P19(Arf), P53, P21(Cip1/Waf1) mRNA were detected by reverse transcription (RT)-PCR. The purity of separated Sca-1(+) HSC was 87.2% and the survival of Sca-1(+) HSC was 96~99%. After 6 h cocultured with 100 μmol/L t-BHP, the ability of aging Sca-1(+) HSC to form mixed hematopoietic progenitor colony, self-renewal and multi-differentiation were decreased significantly. The number of aging Sca-1(+) HSC entered G1 phase of the cell cycle and the percentage of positive cells expressed SA-β-gal increased significantly. The telomere length shortened and the telomerase activity decreased. The expression of p16(INK4a), p19(Arf), p53, P21(Cip1/Waf1) mRNA increased. The t-BHP can induce Sca-1(+) HSC senescence in vitro. The signal transduction pathways of p16(INK4a)-retinoblastoma and P19(Arf)-Mdm2-P53-P21(Cip1/Waf1) may play key roles in the Sca-1(+) HSC senescence induced by t-BHP.
本研究旨在建立体外小鼠造血干细胞（HSC）衰老模型，并探讨其相关的生物学机制，旨在为寻找延缓 HSC 衰老的方法奠定基础。采用免疫磁珠分离法从小鼠骨髓单个核细胞中分离纯化 Sca-1（+）HSC，流式细胞术（FCM）分析分离细胞的纯度，免疫荧光法检测 Sca-1 的表达。采用叔丁基过氧化氢（t-BHP，终浓度 100μmol/L）诱导 6 h 建立体外小鼠 HSC 衰老模型。体外混合造血祖细胞培养、细胞周期检测和衰老相关β-半乳糖苷酶（SA-β-gal）细胞化学染色评估衰老 HSC 的生物学特性。Southern 印迹和端粒重复扩增探针聚合酶链反应（TRAP-PCR）扩增检测端粒长度和端粒酶活性。逆转录（RT）-PCR 检测 p16（INK4a）、P19（Arf）、P53、P21（Cip1/Waf1）mRNA 的表达。分离的 Sca-1（+）HSC 的纯度为 87.2%，Sca-1（+）HSC 的存活率为 96%~99%。与 100μmol/L t-BHP 共培养 6 h 后，衰老 Sca-1（+）HSC 形成混合造血祖细胞集落、自我更新和多向分化的能力明显下降。衰老 Sca-1（+）HSC 进入细胞周期 G1 期的细胞数量增加，阳性细胞表达 SA-β-gal 的比例明显增加。端粒长度缩短，端粒酶活性降低。p16（INK4a）、p19（Arf）、p53、P21（Cip1/Waf1）mRNA 的表达增加。t-BHP 可诱导体外 Sca-1（+）HSC 衰老。p16（INK4a）-视网膜母细胞瘤和 P19（Arf）-Mdm2-P53-P21（Cip1/Waf1）信号转导通路可能在 t-BHP 诱导的 Sca-1（+）HSC 衰老中起关键作用。
