三丁基过氧化氢诱导Sca-1＋造血干／祖细胞衰老与P16INK4a表达的关系  

Relationship between Sca-1^＋ hematopoietic stem and progenitor cells senescence induced by tert-butylhydroperoxide and the expression of P16^INK4a

作　　者：周明[1] 姜蓉[1] 杨斌[1] 姚欣[1] 王亚平[1] 

机构地区：[1]重庆医科大学干细胞与组织工程研究室,组织学与胚胎学教研室,重庆400016

出　　处：《解剖学杂志》2011年第2期172-175,共4页Chinese Journal of Anatomy

基　　金：国家自然科学基金（30973818）;重庆市科委自然科学基金重点项目（CSTC,2009BA5038）

摘　　要：目的：探讨三丁基过氧化氢（t-BHP）诱导Sca-1＋造血干/祖细胞（HSC/HPC）衰老与调控P16INK4a表达的关系,为寻找延缓HSC/HPC衰老方法提供理论和实验依据.方法：免疫磁性分选法分离纯化小鼠Sca-1＋ HSC/HPC.用t-BHP诱导Sca-1＋ HSC/HPC6h建立体外细胞衰老模型,通过衰老相关β-半乳糖苷酶（SA-β-Gal）染色、细胞周期测定和造血祖细胞混合集落（CFU-Mix）培养观察t-BHP诱导Sca-1＋ HSC/HPC衰老的生物学作用;逆转录聚合酶链反应检测衰老相关基因p16INK4a mRNA的表达;蛋白免疫印迹检测P16INK4a蛋白的表达.结果：免疫磁性分选法分离纯化的Sca-1＋ HSC/HPC纯度可达87.33%±1.25%.衰老组细胞SA-β-Gal染色阳性率和G1期细胞比例高于对照组,生成CFU-Mix数低于对照组,衰老组P16INK4a mRNA及蛋白的表达水平较对照组上调.结论：t-BHP能有效诱导Sca-1＋ HSC/HPC衰老,其机制可能与调节P16INK4a mRNA及蛋白的表达有关.Objective： To investigate the relationship between Sca-1^＋ hematopoietic stem and progenitor cells （HSC/HPC） senescence induced by tert-butylhydroperoxide （t-BHP） and the expression of P16INK4a And to build the foundation for searching the methods of how to delaying HSC senescence. Methods： Sca-1^＋ HSC/HPC was isolated and purified by magnetic activated cell sorting （MACS）. The Sca-1^＋ HSC/HPC was induced by t-BHP （final concentration was 100/μmol/L） for 6 h to establish the HSC/HPC aging model in vitro. The changes of cells observed by senescence-associated β-galactosidase （SA-β-Gal） staining, cell cycle assay and culture of mixed hematopoietic progenitor cell （CFU-Mix） were used to investigate the effect of t-BHP on Sca-1^＋ HSC/HPC senescence. The expressions of P16^INK4a mRNA and protein were detected by reverse transcription polymerase chain reaction （RT-PCR） and Western blotting. Results： The purity of separated Sca-1^＋ HSC/HPC was 87. 330% ±1.25%. After 6-hour co-culture with 100 μmol/L t-BHP, the percentage of positive cells expressed SA-β-Gal and the number of cells entered G1 phase in the aged group was higher than that in the control group, while the number of CFU-Mix was lower than that in the control group. Compared with the control group, the expression of P16^INK4a mRNA and protein was up-regulated in the aged group. Conclusion： t-BHP can induce Sca-1^＋ HSC/HPC senescence in vitro. The possible mechanism is that t-BHP can up-regulated the expression of P16^INK4a mRNA and protein.

关 键 词：造血干/祖细胞 衰老 三丁基过氧化氢 P16^INK4A 

分 类 号：R592[医药卫生—老年医学]