关键词: 骨髓造血细胞, 骨髓基质细胞, 衰老生物学, 酶联免疫吸附测定法, 大鼠

Abstract:

Objective To investigate the effect of senescent bone marrow stromal cells (BMSCs) on aging of hematopoietic cells. Methods The BMSCs were isolated by whole bone marrow adherent culture from the rat, and passaged to 3rd generation (P3). The P3 generation of BMSCs was collected and divided into two groups. The control group was cultured as routine and the aging group was cultured with additional 30g/L D-Galactose (D-Gal) for 48 hours. And then, the amounts of granulocyte macrophage colony-stimulating factor(GM-CSF), stem cell factor(SCF), interfeukin(IL)-6, IL-1β，IL-2 and tumor necrosis factor-α(TNF-α) in BMSCs culture supernatant were assayed by ELISA; the proliferation ability of BMSCs was detected by cell counting Kit-8(CCK-8); the senescenceassociated β-galactosidase（SA-β-Gal）staining was used to detect the senescent BMSCs. Bone marrow mononuclear cells(BMMNCs) from rat femur marrow were isolated and purified. Co-culturing aging BMSCs with BMMNCs for 24 hours, and then BMMNCs was collected. The proliferation ability of BMMNCs were measured by CCK-8; the proliferation and differentiation ability of CFU-Mix was detected by colony forming assay; the SA-β-Gal staining was used to detect the senescent BMMNCs; the cell cycle distribution and the ratio of apoptosis of BMMNCs were analyzed by flow cytometry (FCM); DCFH-DA fluorescent staining and FCM were analyzed the level of reactive oxygen species (ROS) in BMMNCs. Malondialdehyde (MDA) content and total superoxide dismutase (SOD) activity were analyzed using enzymatic assay. Results The proliferation of aging group BMSCs was significantly decreased. The positive ratio of SA-β-Gal was markedly increased. The amount of GM-CSF, SCF, IL-6, IL-1β in BMSCs culture supernatant of aging group were obviously decreased and the level of IL-2、TNF-α were raised. After co-cultured BMMNCs with aged BMSCs for 24 hours, the positive ratio of SA-β-Gal stained BMMNCs was significantly increased; the proliferation ability of BMMNCs were declined; the number of CFU-Mix formation was significantly decreased; the BMMNCs were held in G1 phase, and the apoptosis rate was increased; the level of ROS and MDA in BMMNCs was significantly increased and total SOD activity was inhibited. Conclusion D-Galactose may establish BMSCs aging in vitro. The senescence of BMSCs may lead to the hematopoietic cells dysfunction, and the mechanism may be related to the effect of oxidative damage.

Key words: Bone marrow hematopoietic cell, Bone marrow stromal cell, Senescence biology, Enzyme linked immunosorbent assay, Rat