三丁基过氧化氢通过调控细胞周期诱导Sca-1^+造血干/祖细胞衰老  

Tert-butylhydroperoxide induces aging of Sca-1^+ hematopoietic stem and progenitor cells by regulating cell cycle

作　　者：周玥[1] 姜蓉[1] 王萍[1] 杨斌[1] 姚欣[1] 刘典锋[1] 王亚平[1] 

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

出　　处：《第三军医大学学报》2011年第18期1893-1896,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30973818);重庆市自然科学基金(CSTC2009BA5038)~~

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)衰老的细胞周期调控机制。方法免疫磁性分选法分离纯化小鼠Sca-1+HSC/HPC后分为对照组(常规培养细胞),衰老模型组(用t-BHP复制细胞体外衰老模型)。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;RT-PCR检测衰老相关基因p16INK4a、p19Arf、p53、p21Cip1/Waf1 mRNA的表达。Western blot检测细胞周期调控蛋白P16INK4a、P21Cip1/Waf1、CyclinD1、Cyclin E、CDK4、CDK2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染色阳性率为(57.9±4.2)%,高于对照组[(9.1±2.4)%,P<0.05],G0/G1期细胞比例为(87.53±4.03)%,高于对照组[(72.26±3.40)%,P<0.05],生成造血祖细胞混合集落(CFU-Mix)数为(1.5±1.2)/104,低于对照组[(9.8±2.1)/104,P<0.05]。与对照组比较,衰老组细胞p16INK4a、p19Arf、p53、p21Cip1/Waf1 mRNA及P16INK4a、P21Cip1/Waf1、CyclinD1蛋白表达上调(P<0.05),Cyclin E、CDK4、CDK2蛋白表达下调(P<0.05)。结论 t-BHP能有效诱导Sca-1+HSC/HPC衰老,其机制可能与调控细胞周期调控分子的表达有关。Objective To study the cell cycle regulation mechanism underlying tert-butylhydroperoxide（t-BHP）-induced aging of Sca-1＋ hematopoietic stem and progenitor cells（HSC/HPC）.Methods Sca-1＋HSC/HPC were isolated and purified by magnetic activated cell sorting（MACS） and divided into control group and aging model group.Sca-1＋HSC/HPC in control group were routinely cultured.An in vitro aging model of Sca-1＋HSC/HPC was induced by t-BHP.Biological role of t-BHP in inducing aging of Sca-1＋HSC/HPC was observed with aging-associated β-galactosidase（SA-β-gal） staining,cell cycle assay and culture of colony-forming units in mixed HPC.Expressions of aging-related p16INK4a,p19Arf,p53,p21Cip1/Waf1mRNA and P16INK4a,P21Cip1/Waf1,CyclinD1,CyclinE,CDK2 and CDK4 protein were detected by reverse transcription polymerase chain reaction（RT-PCR） and Western blotting,respectively.Results The purity of Sca-1＋ HSC/HPC isolated by MACS was 87.33%±1.25% in aging model group.The positive staining rate and the ratio of Sca-1＋ HSC/HPC were higher in aging model group than in control group（57.9%±4.2% vs 9.1%±4.2%,87.53%±4.03% vs 72.26%±3.4%,P0.05）.The number of colony-forming units in mixed HPC was less in aging model group than in control group [（1.5±1.2）/104 vs（9.8±2.1）/104,P0.05].The expression levels of p16INK4a,p19Arf,p53,p21Cip1/Waf1mRNA,and P16INK4a,P21Cip1/Waf1,CyclinD1 protein were higher while those of Cyclin E,CDK4,CDK2 protein were lower in aging model group than in control group（P0.05）.Conclusion t-BHP can effectively induce the aging of Sca-1＋ HSC/HPC by regulating the expression of cell cycle regulatory molecules.

关 键 词：造血干/祖细胞 衰老 三丁基过氧化氢 细胞周期调控分子 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学] R339.38[医药卫生—基础医学]