Overexpression PSMB5 Prevents Senescence Of Human Bone Marrow Stromal Cells In Vitro

Objective:To observe the role of 20 S proteasome subunit beta 5(PSMB5) in prevents senescence of human bone marrow stromal cells(h BMSCs) in vitro, and confirm the role of the 20 S proteasome activity changing in the aging process, which would further looking out a feasible strategy to investigate the prevention of cell replicative senescence.Methods:The human BMSCs were infected by PSMB5 lentiviral vectors with Green Fluorescent Protein(GFP). Cells were divided into PSMB5 Group and GFP Control Group according to different virus infection. 1. The expression of PSMB5 was detected by Western blotting, and Real-time PCR also was used to validation the expression of 20 S proteasome subunit. 2. The activity of 20 S proteasome was assessed by proteasome activity assay. 3. Bromodeoxyuridine(Brd U) assay was used to observe the proliferation of cells in two groups. Through the Western to detect the cell cycle-related proteins: P21, Cyclin D1 and CDK4 were detected by Western blotting. 4. To investigate the influence of niche on h BMSCs aging process, senescenceassociated-β-galactosidase(SA-β-gal) staining were performed to identify senescent cells in the forebrain. 5. The h BMSCs were exposed to H2O2 in different concentrations(0 μM, 50 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM) at the specified concentrations for 30 min, followed by assessment of viability using the CCK-8 assay.Results:1. Western blotting showed that the expression of PSMB5 was about 2.70±0.19 times(P<0.01) in the PSMB5 group compared with the GFP group. The real-time PCR showed that the expression of PSMB5 m RNA for h BMSCs in the PSMB5 group wereraised almost 5.24±0.22 times(P<0.01), compared to the early-stage. But there was no statistical significance about the expression of others as PSMB1, PSMB2, PSMB6. The 20 S proteasomal activity was increased by 1.55±0.21(P<0.05). 3. Brd U incorporation showed that the positive rate of Brd U positive cells was within(37.91%±1.82%) in the PSMB5 group, and markedly higher than(28.18%±1.98%), the rate in the GFP group(P<0.05).Moreover, the result of Western blotting found that in PSMB5 group the expression of cell cycle-related proteins P21 increased to 1.45±0.05 times(P<0.01) compared to GFP group, the Cyclin D1 raised about 1.56±0.16 times(P<0.01), the CDK4 raised about 1.23±0.06 times(P<0.05). 4. SA-β-gal staining were used to detect senescence cells, few positive cells were found in PSMB5 group, the rate within(40%±3.7%) compared to GFP group(55%±3%)(P<0.01). 5. The CCK-8 assay results showed that cell viability was decreased in a concentration-dependent manner after incubation with H2O2, cell activity will decreased with H2O2 incubation concentration reduced(P<0.01). The PSMB5 group have more cell activity in the same H2O2 incubation concentration, and the ratio of two groups was 2.11±0.23 times(P<0.01) when at the 300 μM of H2O2 incubation concentration, but there was no statistical significance at the 300 μM of concentration. The results of 20 S proteasome activity showed that the PSMB5 group is 1.37±0.12 times(P<0.01) compared with the GFP group. The results showed that in conditions of oxidative stress, overexpression of PSMB5 can promote the cell cycle, protect the cell proliferative potential, increase cell viability.Conclusion:1. The 20 S proteasome activity and the expression of PMSB5 were increased after PSMB5 gene Overexpression and the expression of cell cycle-related proteins and the proliferation of h BMSCs increased at the same time. 2. The increased expression of PMSB5 can be improved by the proteasome activity to protect cells from oxidative stress causing damage to cells, may be an important target for inhibition of cell aging and improving the viability of cells.