骨髓基质细胞衰老对造血细胞氧化应激的影响

发布时间：2019-01-20 15:32

【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。
[Abstract]:Objective to investigate the effect of bone marrow stromal cell aging on oxidative stress of bone marrow hematopoietic cells. Methods the aging model of bone marrow stromal cells (BMSCs) was established by whole-bone marrow adherent culture (BMSCs),) in male healthy C57 mice at the age of 6 ~ 8 weeks. The experiment was divided into routine control group and aging group. The control group was cultured in conventional culture medium for 48 hours, and the aging group was treated with 30g/L D galactose for 48 h. Senescence associated 尾 -galactosidase (SA- 尾 -galactosidase) staining was used to detect the percentage of aging bone marrow stromal cells and flow cytometry was used to analyze the cell cycle. Bone marrow mononuclear cells (BMNCs) were co-cultured with bone marrow-based cells (BMC), trypanosome blue count (BMNCs) was counted, bone marrow cell cycle was detected by flow cytometry (FCM), myeloid multidirectional hematopoietic progenitor cells (CFU-Mix) semisolid culture colony count was detected. The levels of reactive oxygen species (BMNCs) and reactive oxygen species (ROS) in bone marrow cells and bone marrow cells were detected by DCFH-DA fluorescence staining and laser scanning confocal microscopy. The expression of connectin 43 (Cx43) in bone marrow-based cells was detected by laser scanning confocal microscopy. Results Dgalactose induced the senescence of bone marrow-based cells in vitro, and the positive rate of SA- 尾 Gal staining in bone marrow cells increased significantly, and the G1 phase of cell cycle was blocked. After co-culture of BMNCs and aging group for 48 h, the number of BMNCs living cells counted by trypanosome blue decreased. Cell cycle arrest and CFU-Mix colony formation decreased significantly compared with the control group. In aging group, the content of ROS increased and the expression of Cx43 was down-regulated, and the content of ROS in BMNCs co-cultured with senescent BMSCs was higher than that in BMNCs co-cultured with normal control. Conclusion the aging bone marrow stromal cells inhibit the proliferation and differentiation of bone marrow hematopoietic cells. The mechanism may be related to the enhancement of oxidative stress of aging bone marrow stromal cells and the reduction of oxidative stress ability of hematopoietic cells.
【作者单位】：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
【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)
【分类号】：R329.2

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