健康人骨髓CD34~+造血干/祖细胞衰老与年龄的相关性研究
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摘要
目的探讨增龄过程中健康人CD34~+造血干/祖细胞(CD34~+HSC/HPCs)衰老的变化过程。方法由重庆医科大学附属第一医院血液科骨穿室提供正常人骨髓9例,将其分为青年组3例(女性31岁、男性26岁、男性28岁)、中年组3例(男性59岁、男性57岁、女性52岁)和老年组3例(男性70岁、男性70岁、女性62岁)。提取骨髓单个核细胞(BMNCs)后,免疫磁性细胞分选法分离纯化CD34~+细胞群,流式细胞术检测细胞纯度,台盼蓝染色检测细胞活性,衰老相关β-半乳糖苷酶(SA-β-gal)染色观察各年龄组阳性细胞百分比;造血干/祖细胞混合集落培养比较各年龄组CD34~+造血干/祖细胞形成混合集落能力。结果分选前每106个BMNCs中CD34~+细胞群比例为(1. 31±0. 54)%;免疫磁性分选后每106个细胞中CD34~+细胞群比例为(92. 5±0. 39)%。各年龄组人骨髓CD34~+造血干/祖细胞存活率可达99. 1%以上。人骨髓CD34~+造血干/祖细胞的SA-β-gal染色阳性率出现明显的随增龄变化趋势,老年组明显高于中年组及青年组。CD34~+造血干/祖细胞形成的干细胞混合集落能力随年龄增加降低。结论随年龄增加人骨髓CD34~+造血干/祖细胞出现明显衰老,表现为造血增殖能力下降,可能是造成一些老年人血液学疾病的原因。
Objective To investigate the senescence process of CD34~+hematopoietic stem/progenitor cells( CD34~+HSC/HPCs) in healthy human aging. Methods The human bone marrow without significant hematological abnormalities collected from 9 healthy people who were in hematology department of first affiliated hospital of Chongqing medical university were divided into three groups: youth group( 3 cases: female 31 years old,male 26 years old,male 28 years old),middle age group( 3 cases: male 59 years,male 57 years,female 52 years old),the elderly age group( 3 cases: male 70 years old,male 70 years old,female 62 years old). CD34~+cells were separated and purified by immunomagnetic adsorption cell sorting( MACS) from BMNCs. Cell purity was detected by flow cytometry. Cell viability was assayed by trypan blue staining. Senescence-associated β-galactosidase( SA-β-gal) staining was used to observe the percentages of positive cells in all groups. Hematopoietic stem/progenitor cells mixed colony forming unit( CFU-Mix) culture was used to evaluate the colony forming ability of CD34~+CD34~+HSC/HPCs of all groups. Results The proportion of CD34~+cells per 106 BMNCs was( 1. 31 ± 0. 54) % before sorting and( 92. 5 ± 0. 39) % per 106 cells after immunomagnetic sorting. The survival rate of CD34~+HSC/HPCs in human bone marrow was 99. 1% in all groups. The positive rate of SA-β-gal staining of CD34~+HSC/HPCs in human bone marrow of elder group was significantly higher than that in middle-aged group or youth group. The number of CFU-Mix formed by CD34~+HSC/HPCs in young,middle and elderly patients decreased,and the number of cells in colonies decreased. Conclusion CD34~+hematopoietic stem/progenitor cells in human bone marrow show obviously senescent changes during aging,which may be the cause of some hematological diseases in the old human.
Objective To investigate the senescence process of CD34~+hematopoietic stem/progenitor cells( CD34~+HSC/HPCs) in healthy human aging. Methods The human bone marrow without significant hematological abnormalities collected from 9 healthy people who were in hematology department of first affiliated hospital of Chongqing medical university were divided into three groups: youth group( 3 cases: female 31 years old,male 26 years old,male 28 years old),middle age group( 3 cases: male 59 years,male 57 years,female 52 years old),the elderly age group( 3 cases: male 70 years old,male 70 years old,female 62 years old). CD34~+cells were separated and purified by immunomagnetic adsorption cell sorting( MACS) from BMNCs. Cell purity was detected by flow cytometry. Cell viability was assayed by trypan blue staining. Senescence-associated β-galactosidase( SA-β-gal) staining was used to observe the percentages of positive cells in all groups. Hematopoietic stem/progenitor cells mixed colony forming unit( CFU-Mix) culture was used to evaluate the colony forming ability of CD34~+CD34~+HSC/HPCs of all groups. Results The proportion of CD34~+cells per 106 BMNCs was( 1. 31 ± 0. 54) % before sorting and( 92. 5 ± 0. 39) % per 106 cells after immunomagnetic sorting. The survival rate of CD34~+HSC/HPCs in human bone marrow was 99. 1% in all groups. The positive rate of SA-β-gal staining of CD34~+HSC/HPCs in human bone marrow of elder group was significantly higher than that in middle-aged group or youth group. The number of CFU-Mix formed by CD34~+HSC/HPCs in young,middle and elderly patients decreased,and the number of cells in colonies decreased. Conclusion CD34~+hematopoietic stem/progenitor cells in human bone marrow show obviously senescent changes during aging,which may be the cause of some hematological diseases in the old human.
引文
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[2]周玥,杨斌,王亚平.人参皂苷Rg1延缓造血干细胞衰老与P16INK4a表达关系的研究[J].中国中药杂志,2011,36(5):1-6.doi:10.4268/cjcmm20110520.
[3]耿珊,张琛,王亚平,等.过氧化损伤与抗氧化能力时间动态变化与造血干/祖细胞衰老的相关性研究[J].中国老年学杂志,2013,33(5):1061-1063.doi:10.3969/j.issn.1005-9202.2013.37.0.
[4]Zhou Y,Yang B,Yao X,et al.Establishment of an aging model of Sca-1+hematopoietic stem cell and studies on its relative biological mechanisms[J].In Vitro Cell Dev Biol Anim,2011,47(2):149-156.doi:10.1007/s11626-010-9372-5.
[5]Geng S,Mu XY,Chen XB,et al.Study on the dynamic biological characteristics of Sca-1+Hematopoietic stem and progenitor cell senescence[J].Stem Cells Int,2015,2015:954120.doi:10.1155/2015/954120.
[6]周玥,杨斌,王亚平.人参皂苷Rg1延缓造血干细胞衰老及其相关机制[J].中华医学杂志,2010,90(48):3421-3425.doi:10.3760/cma.j.issn.0376-2471.2010.48.010.
[7]周玥,杨斌,王亚平.小鼠造血干细胞体外衰老模型的构建及其相关生物学[J].解剖学报,2010,41(5):699-705.doi:10.3969/j.issn.0529-1356.2010.05.010.
[8]周玥,杨斌,王亚平.Sca-1+造血干/祖细胞重建致死剂量辐射小鼠造血功能的作用[J].中国组织工程研究与临床康复,2010,14(32):605-609.doi:10.3969/j.issn.1673-8225.2010.
[9]周玥,姜蓉,王亚平.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[J].解剖学杂志,2011,34(2):172-175.doi:10.3969/j.issn.1001-1633.2011.02.009.
[10]Chen C,Mu X,Zhou Y,et al.Ginsenoside Rg1 enhances the resistance of hematopoietic stem/progenitor cells to radiation-induced aging in mice[J].Acta Pharmacologica Sinica,2014,35(1):143-150.doi:10.1038/aps.2013.136.
[11]Hu W,Jing P,Wang L,et al.The positive effects of Ginsenoside Rg1upon the hematopoietic microenvironment in a D-Galactose-induced aged rat model[J].BMC Complement Altern Med,2015,15:119.doi:10.1186/s12906-015-0642-3.
[12]Boehme KA,Schleicher SB,Traub F,et al.Chondrosarcoma:a rare misfortune in aging human cartilage?The role of stem and progenitor cells in proliferation,malignant degeneration and therapeutic tesistance[J].Int J Mol Sci,2018,19(1):311.doi:10.3390/ijms19010311.
[13]Janzen V,Forkert R,Fleming HE,et al.Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a[J].Nature,2006,443(7110):421-426.doi:10.1038/nature05159.
[14]Rossi DJ,Jamieson CH,Weissman IL,et al.Stems cells and the pathways to aging and cancer[J].Cell,2008,132(4):681-696.doi:10.1016/j.cell.2008.01.036.
[15]Vas V,Senger K,D9rr K,et al.Aging of the microenvironment influences clonality in hematopoiesis[J].PLo S One,2012,7(8):e42080.doi:10.1371/journal.pone.0042080.
[16]Ju Z,Jiang H,Jaworski M,et al.Telomere dysfunction induces environmental alterations limiting hematopoietic stem cell function and engraftment[J].Nat Med,2007,13(6):742-747.doi:10.1038/nm1578.
[17]Henry CJ,Marusyk A,Zaberezhnyy V,et al.Declining lymphoid progenitor fitness promotes aging-associated leukemogenesis[J].Proc Natl Acad Sci USA,2010,107(50):21713-21718.doi:10.1073/pnas.1005486107.
[18]Velarde MC,Demaria M,Campisi J.Senescent cells and their secretory phenotype as targets for cancer therapy[J].Interdiscip Top Gerontol,2013,38:17-27.doi:10.1159/000343572.
[19]Kang TW,Yevsa T,Woller N,et al.Senescence surveillance of pre-malignant hepatocytes limits liver cancer development[J].Nature,2011,479(7473):547-551.doi:10.1038/nature10599.
[20]Roobrouck VD,Ulloa-Montoya F,Verfaillie CM,et al.Self-renewal and differentiation capacity of young and aged stem cells[J].Exp Cell Res,2008,314(9):1937-1944.doi:10.1016/j.cell.2008.01.036.
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[23]Pearce D,Bonnet D.Ageing within the hematopoietic stem cell compartment[J].Mech Ageing Dev,2009,130(1-2):54-57.doi:10.1016/j.mad.2008.04.005.
[24]Pang WW,Price EA,Sahoo D,et al.Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age[J].Proc Natl Acad Sci USA,2011,108(50):20012-20017.doi:10.1073/pnas.1116110108.
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[26]Li J,Cai D,Yao X,et al.Protective effect of ginsenoside Rg1 on hematopoietic stem/progenitor cells through attenuating oxidative stress and the Wnt/β-catenin signaling pathway in a mouse model of d-galactoseinduced aging[J].Int J Mol Sci,2016,17(6):849.doi:10.3390/ijms17060849.
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[28]Xiao Y,Wang J,Song H,et al.CD34+cells from patients with myelodysplastic syndrome present different p21 dependent premature senescence[J].Leuk Res,2013,37(3):333-340.doi:10.1016/j.leukres.2012.11.006.
[29]Wang ZL,Chen LB,Qiu Z,et al.GinsenosideRg1 ameliorates testicular senescence changes in dgalinduced aging mice via antiinflammatory and antioxidative mechanisms[J].Mol Med Rep,2018,17(5):6269-6276.doi:10.3892/mmr.2018.8659.(编辑:左艳芳)