骨髓基质细胞衰老对造血细胞氧化应激的影响  

Effects of senesence of bone marrow stromal cells on hematopoietic cells oxidative stress

作　　者：宋小英[1] 景鹏伟 熊丽溶 贾道勇 王亚平[1] 王璐[1] 

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

出　　处：《解剖学报》2015年第6期757-763,共7页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(81173398);重庆市科委基础与前沿研究资助项目(cstc2014jcyj A10001)

摘　　要：目的探讨骨髓基质细胞衰老对骨髓造血细胞氧化应激的影响。方法取6-8周雄性健康C57小鼠,全骨髓贴壁法培养骨髓基质细胞（BMSCs）,建立衰老骨髓基质细胞体外模型。实验分为常规对照组和衰老组。对照组：常规培养基培养48h;衰老组：常规培养基内加入30g/L D-半乳糖,作用48h。衰老相关β-半乳糖苷酶（SA-β-Gal）染色检测衰老骨髓基质细胞百分率;流式细胞术分析骨髓基细胞周期。骨髓单个核细胞（BMNCs）与骨髓基细胞共培养,锥虫蓝计数BMNCs活细胞数;流式细胞术检测骨髓基细胞周期;髓系多向性造血祖细胞（CFU-Mix）半固体培养集落计数;DCFH-DA荧光染色流式检测BMNCs和骨髓基细胞的活性氧簇（ROS）水平;激光扫描共焦显微术半定量检测骨髓基细胞连接蛋白43（Cx43）表达。结果 D-半乳糖体外诱导骨髓基细胞衰老,骨髓基细胞SA-β-Gal染色阳性率显著上升,细胞周期G1期阻滞。BMNCs与衰老组骨髓基细胞共培养48h,锥虫蓝计数BMNCs活细胞数量下降;细胞周期阻滞;CFU-Mix集落形成数量较对照组显著下降。衰老组骨髓基细胞胞内ROS含量增加,Cx43表达明显下调;与衰老骨髓基细胞共培养的BMNCs胞内ROS含量较与常规对照骨髓基细胞共培养的BMNCs胞内ROS含量上升。结论衰老骨髓基质细胞抑制骨髓造血细胞增殖分化,其机制可能与衰老骨髓基质细胞氧化应激增强,缓解造血细胞氧化应激能力下降有关。Objective To explore the impact of oxidative stress between aging bone marrow stromal cells and hematopoietic cells and to provide the theoretic and experimental evidences for explaining the effect of senescence hematopoietic inductive microenvironment （HIM） on proliferation and differentiation of hematopoietic cells and its possible maehanism. Methods Bone marrow stromal cells were isolated by whole bone marrow adherent culture from healthy male C57 mice about 6-8 weeks. The cells were divided into two groups ： the control group and the aging group was cultured with additional 30g/L D-galactose （D-Gal） for 48 hours. The ratio of the SA-β-Gal staining positive bone marrow stromal cells was counted; the cell cycle distribution of bone marrow stromal ceils was analyzed by flow cytometry （FCM）. The bone marrow mononuclear cells （BMNCs） extracted from femur bone marrow were co-cultured with bone marrow stromal cells. The number of BMNCs was counted by trypan blue staining; the cell cycle distribution Of BMNCs was analyzed by flow cytometry. The myeloid hematopoietic progenitor cells （CFU-Mix） were cultured by semi-solid cultivation assay and the number of forming colonies was counted. Flow cytometric DCFH-DA fluorescent staining analyzed the level of reactive oxygen species（ROS） in bone marrow stromal cells and BMNCs; bone marrow stromal cells protein expression of connexin- 43 （Cx43） was detected by immunofluoescence assay. Results The positive ratio of SA-β-Gal was significantly increased in the aging bone marrow stromal ceils . The bone marrow stromal cells were hold in G1 phase arrest. After co-cultured BMNCs with bone marrow stromal cells for 48hous, the number of live BMNCs went decreased; the cell cycle of BMNCs was arrested; the number of CFU-Mix forming colonies was significantly decreased compared with the control group. The ROS level of bone marrow stromal cells was significantly increased in the aging group. The expression of proteins Cx43 was obviously down-regulated in th

关 键 词：衰老 骨髓基质细胞 造血细胞 氧化应激 流式细胞术 小鼠 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]