Abstract: Objective To establish the aging model of rat bone marrow stromal cells (BMSCs) in vitro and in vivo, in order to study the senescence biology of aging BMSCs. Methods The control cell group (in vitro): isolating, purifying and culturing BMSCs from healthy male SD rats. collecting the third generation (P3) of BMSCs for analysis. The aging model group (in vitro): the P3 BMSCs was co-cultured with D-galactose (D-Gal, 30 g/L) for 48 hours. The aging rat model group (in vivo): the rat were given 120 mg D-Gal by the way of daily neck subcutaneous injection for 42 consecutive days. The control rat group (in vivo): the rats were administrated with the same volume of saline for the same times. On the second day after the aging model was established, the BMSCs were collecting and culturing for study. 1)The proliferative potency was detected by Cell Counting Kit-8(CCK-8); the distribution of cell cycle and apoptosis by flow cytometry (FCM); 2)the ratio of aging BMSCs by the senescence-associated β-galactosidase(SA-β-Gal) staining; 3)malonaldehyde(MDA) content and total superoxide dismutase(SOD) activity by enzymatic assay; the level of reactive oxygen species (ROS) by DCFH-DA fluorescent staining counted with FCM; 4)the expression level of senescence-related signaling proteins of P16,P21,P53,CDK2 and cyclin-D by Western blotting. Results Compared with the matched control group, the BMSCs of aging model group displayed a significant decrease in proliferation; the BMSCs were held in G1 phase arrest as the proportion of the cells in G1 phase increased, while that decreased in S phase(P<0.05); and the positive ratio of SA-β-Gal stained BMSCs also significantly increased(P<0.05); BMSCs in the aging model group showed an increasing level of ROS and MDA, meanwhile a decline in total SOD activity was decreased(P<0.05);P16,P21 and P53 protein expression in aging BMSCs was obviously enhanced(P<0.05), at the same time the expression of CDK2 and cyclin-D was also decreased(P<0.05). Conclusions D-Gal can be used to build the aging model of BMSCs equally in vitro and vivo, It acts through up-regulation of expressions of aging-related proteins and inhibition of level of oxidative stress injury and chronic inflammation.

Key words: bone marrow stromal cell, D-Gal, senescence biology, rat, mechanism