关键词: Hematopoietic stem cell, Aging, Model EM>in vitro/EM>, Telomere, Telomerase, SA-β-gal staining, Flow cytometry, RT-PCR, Mouse

Abstract: 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-1SUP>+/SUP>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-1SUP>+/SUP> antigen was detected by immunofluorescence. Sca-1SUP>+/SUP>HSCs were induced by tertbutylhydroperoxide( t-BHP, final concentration of 100 μmol/L) for 6 hours to establish the HSCs aging model EM>in vitro/EM>. Biological characteristics of aging HSCs was evaluated by mixed hematopoietic progenitor cell culture, cell cycle assay and sensscence-associated β-galactosidase (SA-β-gal) cytochemical 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 p16SUP>Ink4a/SUP>, p19SUP>Arf/SUP>, p53, p21SUP>Cip1/Waf1/SUP>mRNA was detected. Results The purity of separated Sca-1SUP>+ /SUP>HSCs was （87.33±1.25）%. After being cultured with 100 μmol/L t-BHP for six hours, the ability of aging Sca-1SUP>+/SUP> HSCs to forming mixed hematopoietic progenitor colony, self-renewal and multidifferentiation was decreased significantly. The number of aging Sca-1SUP>+/SUP> HSCs entered GSUB>1/SUB> phase of the cell cycle, the percentage of SA-β-gal positive cells and the expression of p16SUP>Ink4a/SUP>, p19SUP>Arf/SUP>, p53, p21SUP>Cip1/Waf1/SUP>mRNA were increased. The telomere length was shorten and the telomerase activity was

Key words: 造血干细胞, 衰老, 体外模型, 端粒, 端粒酶, 衰老相关β-半乳糖苷酶染色, 流式细胞术, 反转录-聚合酶链反应, 小鼠