氧化低密度脂蛋白通过氧化应激诱导小鼠造血干细胞衰老

 目的 探讨氧化低密度脂蛋白（ox-LDL）体外诱导造血干细胞(HSCs)衰老的可能机制。方法 用免疫磁性分选法分离纯化小鼠HSC，与ox-LDL共培养，采用β-半乳糖苷酶（SA-β-Gal）染色检测衰老HSC，流式细胞术检测HSC细胞周期分布，混合集落培养(CFU-Mix)检测HSC混合集落形成能力。流式细胞术和免疫荧光检测HSC产生活性氧（ROS）的量，酶学比色法检测HSC培养上清液超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）及丙二醛（MDA）含量。Southern blot和TRAP-PCR法检测HSC端粒长度和端粒酶活性。结果 ox-LDL诱导HSC呈现典型的衰老生物学表现：SA-β-Gal染色阳性细胞率显著增高（P <0.01）； G0/G1 期比例明显增加，S 期显著减少（P <0.01） ；CFU-Mix数量显著减少（P <0.01）。衰老HSC端粒缩短（P <0.05），端粒酶活性降低（P <0.05）。衰老HSC ROS含量显著增加（P <0.01），细胞培养上清液中SOD、GSH-Px活力下降、 MDA含量增加（P <0.05）。 结论 ox-LDL能通过氧化应激诱导HSC衰老，其机制可能与ROS的蓄积及抗氧化酶活性受抑引起端粒功能异常有关。

Oxidation low density lipoprotein induces aging of mouse hematopoietic stem cells by oxidative stress

Objective To explore the underlying mechanism that Oxidation low density lipoprotein(ox-LDL) induced aging of hematopoietic stem cells (HSCs) in vitro. Methods Mouse HSCs were isolated by magnetic cell sorting and was cultured with ox-LDL. Cell cycles were detected by flow cytometry and identify aging HSC. CFU-Mix cultivation was used to evaluate the potency of differentiation in HSCs. The production of reactive oxygen specie (ROS )in HSCs was evaluated by flow cytometry analysis and laser scanning confocal microscope assess, respectively. The activities of superoxide dismutase (SOD )and glutathione peroxidase (GSH-Px) and the levels of malondialdehyde（MDA）in supernatant that HSC cultured IMDM were detected by chemical colorimetric method. Senescence-associated β-galactosidase (SA-β-Gal) staining was used to detect aging HSCs. Southern blotting and TRAP-PCR were used to evaluated the length of telomere and the activity of telomerase in HSCs, respectively. Results Exogenous ox-LDL induced HSC aging was compared to HSC without ox-LDL treatment group. Biological feature of aged HSC as follows:1）The percentage of SA-β-Gal positive cells was significantly increased; the ratio of G0/G1 stages was strikingly increased , while the ratio S stage was decreased; the number of CFU-Mix was also notably decreased. 2) The length of telomere was remarkedly Shortening in aged HSC, and the activity of telomerase of aged HSC was also reduced. 3) The content of ROS was increased significantly and the vitality of SOD and GSH-Px was decreased in aged HSC, while the content of MDA in supernatant that aged HSC cultured IMDM was increased . Conclusions ox-LDL could induce mice HSC aging by oxidative stress, which maybe partly ascribed to the length shorter and the activity of telomerase slower.