关键词: 衰老, 造血诱导微环境, 骨髓基质细胞, 造血细胞, 增殖分化

Abstract: Objective: To explore the influence of senescence-bone marrow stromal cells on the proliferation and differentiation of hematopoietic cells and to provide the theoretic and experimental evidences for explaining the effect of senescent hematopoietic microenvironment on Hematopoietic stem/progenitor cells. Methods: The bone marrow stromal cells (BMSCs) were isolated by whole bone marrow adherent culture from rats, the experiment cells were divided into control group and aging group. The control group was cultured as usual and the aging group was cultured with D-galactose (D-Gal) 30mg/mL for 48 hours. And then the proliferation ability of BMSCs was detected by Cell Counting Kit-8(CCK-8); the distribution of cell cycle was analyzed by flow cytometry (FCM); the senescence-associated β-galactosidase（SA-β-Gal）staining was used to detect the senescent BMSCs; Western Blotting examined the expression level of P16, P21and P53. The bone marrow hematopoietic cells were co-cultured with BMSCs, the proliferation and differentiation ability of CFU-Mix was detected by colony forming assay. The amounts of SCF、GM-CSF and IL-1β in BMSCs culture supernatant were detected by ELISA; DCFH-DA fluorescent staining and FCM analyzed the level of ROS (reactive oxygen species) in BMSCs; MDA (malonaldehyde) content and total SOD (superoxide dismutase) activity was analyzed as well using enzymatic assay. Results: Compared with the control group, D-Gal induced senescent BMSCs displayed a decrease in proliferation; the BMSCs were hold in G0/G1 phase arrest(p<0.01); the positive ratio of SA-β-Gal stained BMSCs also significantly increased; The expression of senescence-related proteins including P16、P21 and P53 were obviously up-regulated(p<0.01). The proliferation and differentiation ability of the hematopoietic cells co-cultured with aging BMSCs declined. In the aging BMSCs, ROS and MDA the index of oxidative damage increased and SOD the antioxidant index declined(p<0.01). The amount of IL-1β、GM-CSF、SCF in BMSCs culture supernatant of aging group decreased(p<0.01) .Conclusion: Senescent BMSCs inhibit proliferation and differentiation of the hematopoietic cells and the underling mechanism may be related to the oxidative damage of BMSCs, thus reduced secretion of cytokines by BMSCs.

Key words: senescence, hematopoietic inductive microenvironment, bone marrow stromal cell, hematopoietic cells., proliferation and differentiation