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Characteristics of bone marrow stromal cells biology in aging rats model
JING Peng-wei HU Wen-xu SONG Xiao-ying ZHANG Yan-yan JIA Dao-yong ZHANG Meng-si XIA Jie-yu LI Jing WANG Ya-ping WANG Lu*
Acta Anatomica Sinica ›› 2015, Vol. 46 ›› Issue (1) : 44-50.
Characteristics of bone marrow stromal cells biology in aging rats model
Objective To explore the characteristic of bone marrow stromal cells (BMSCs) biology in aging rats and to provide the theoretic and experimental evidences for explaining the effect of senescence on hematopoietic inductive microenvironment (HIM). Methods Healthy male SD rats were randomized into two groups. The aging model rats were given 120mg D-galactose (D-Gal) by daily neck subcutaneous injection for 42 consecutive days. As a control, rats were administrated with the same volume of saline for the same period. On the second day after the aging model was established, the bone marrow mononuclear cells (BMNCs) were extracted from the femur bone marrow and cultured for CFU-Mix colony forming assay. The BMSCs were isolated by whole bone marrow adherent culture, and passaged to 3rd generation (F3) as well. For the F3 generation BMSCs, the ability of proliferation 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; the amount of interleukin(IL)-6 and stem cell factor (SCF) in BMSCs culture supernatant were detected by ELISA; DCFH-DA fluorescent staining and FCM analyzed the level of reactive oxygen species(ROS) in BMSCs; malonaldehyde(MDA) content and total superoxide dismutase(SOD) activity were analyzed as well using enzymatic assay; Western blotting examined the expression level of senescencerelated proteins including P16, P21and P53. Results Compared with the control group, the capability of mixed colony forming unit (CFU-Mix) of BMNCs in aging model group was obviously attenuated. The results indicated that BMSCs of aging model rats displayed a decrease in proliferation; the BMSCs were held in G1 phase arrest, the proportion of the cells in G0/G1 phase increased, while the proportion in S phase decreased; the positive ratio of SA-β-Gal stained BMSCs also significantly increased; the amount of IL-6 and SCF in BMSCs culture supernatant of aging model group was lower than that of control. Furthermore the BMSCs in aging model rats showed an increase in ROS and MDA level and a decline in total SOD activity. The expression of senescence-related proteins including P16,P21 and P53 were obviously up-regulated. Conclusion BMSCs in aging model rats show senescence-associated biologic changes, and the underlying mechanism may be related to the activation of senescence signaling pathway due to oxidative damage.
Aging / Microenvironment / Bone marrow stromal cell / Western blotting / Rat
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