Expression And Significance Of Silent Mating Type Information Regulation 1 In Premature Senescence Of Bone Marrow Mesenchymal Stem Cells

Objective:To investigate the differential expression and significance of silent mating type information regulation 1 (SIRT1) in premature senescence of bone marrow MSCs induced in microenvironment of senescent nucleus pulposus cells (NPCs).Methods:The isolation and identification of MSCs and NPCs was performed on bone marrow and caudal disc from Sprague Dawley rat. The co-cuLture system was established in the Transwell small chamber. Three groups were designed as follow: group A,MSCs were cultured alone without any intervention; group B, MSCs were co-cultured with NPCs without any intervention; group C, MSCs were co-cultured with the senescent NPCs induced by oxidative stress. At 3,6 and 9 days after co-culture, SIRT1 activity assay kit, real-time fluorescent quantitative polymerase chain reaction and Western blotting were performed to detect the SIRT1 activity, expression and distribution of mRNA and protein of SIRT1 in MSCs in each group. Level of oxidative stress and cell senescence ware also evaluated by detecting the expression of p53, superoxide dismutase(SOD)l and senescence associated β-galactosidase (SA-β-gal) staining.Results:MSCs and NPCs were successfully isolated and identified. The percentages of CD29-positive,CD34-positive, CD45-positive and CD90-positive cells in total MSCs were 95.0%,0.282%,2.29%and 97.8% respectively. NPCs expressed II collagen and aggrecan. After 3 days of co-culture, SIRT1 activity of group C was significantly higher than that in groups A and B (P<0.05), and then decreased. At 9 days the SIRTl activity of group C showing significant difference when compared with groups A and B(P<0.05). As compared with groups A (1.00±0.01,1.00±0.02,1.00±0.01) and B (0.96±0.05,1.03±0.07, 1.03±0.07),the gene expression of SIRTl in group C was significantly increased at 3 and 6 days (1.39±0.08,1.82±0.10), much lower than that at 9 days (0.74±0.11) after co-culture (P<0.05). Compared with groups A and B, the expression of SIRTl protein in group C was decreased significantly at 3,6,9 days(P<0.05).At 9 days, the expression of SOD1, p53 protein and SA-β-gal activity in group C was much higher when compared with group A and B (P<0.05)Conclusions:Premature senescence of MSCs induced by the paracrine effect of senescent NPCs may be associated with the abnormal expression of SIRT1.