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

Abstract:

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 machanism. 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 cells 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 cells . 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 the aging bone marrow stromal cells . The ROS level in BMNCs co-cultured with senescent bone marrow stromal cells was dramatically increased compared with those BMNCs co-cultured with control bone marrow stromal cells . Conclusion Senescent bone marrow stromal cells inhibited proliferation and differentiation of hematopoietic cells. The underlying mechanism may be related to the increased oxidative stress in senescent BMSCs and the decreased ability of relieving the oxidative stress in hematopoietic cells.

Key words: Senescence, Bone marrow stromal cell, Hematopoietic cell, Oxidative stress, Flow cytometry, Mouse