| Objectives With increasing of age, stem cells are also subject to age-associated changes. Recent studies suggest that the cellular extrinsic factors play important role on aging of adult stem cells. However, it is still unclear that the effects of aged cell-extrinsic environment on the aging of mesenchymal stem cells (MSCs) and which factors are involved. In this study, we examined the effects of old rat serum on senescence, proliferation, survival ability of MSCs and the Wnt/β-catenin signaling, and explored the effects and mechanisms of Wnt/β-catenin signaling on senescence, proliferation, survival ability of MSCs. To provide novel strategy and experimental evidence for clinical therapy of stem cells transplantation in old individuals. Methods Extracted and prepared serum from young (8～12 wk) and aged (64～72 wk) SD rats. Isolated and cultured of MSCs from young (8～12 wk) SD rats. Then MSCs were cultured with 20%young rat serum (YRS) or old rat serum (ORS) for 36 h. The senescence-associated changes were examined with SA-β-galactosidase staining and ROS staining. The proliferation ability was tested by MTT assays. To evaluate the oxidative stress tolerance of MSCs was cultured in ORS and YRS, the number of apoptotic cells was determined by AO/EB staining and Hoechest 33342 staining after MSCs was treated with H2O2. To examine the effects of ORS on the Wnt/β-catenin signaling, the level of Wnt 3 a in YRS or ORS was detected with Elisa assay. Immunofluorescence and western blotting were used to detect the expressions of intracellularβ-catenin and GSK-3β. RT-PCR was used to detect the expression of c-myc which is a target gene of Wnt/β-catenin signaling. For further inhibiting the Wnt/β-catenin signaling, the siRNA (si-β-catenin) was designed and synthesized to silenceβ-catenin. For screening effective siRNA against P-catenin, P-catenin expression was detected by RT-PCR, immunofluorescence and western blotting after transfecting the cells using different siRNAs. In order to examine the effects of different concentrations of Wnt 3 a on senescence of the cells, the cells were cultured in YRS containing 10 ng/ml,50 ng/ml and 100 ng/ml Wnt 3a respectively, then the senescence-associated changes were examined with SA-β-galactosidase staining. For further identifying the effects of the Wnt/β-catenin signalling on aging, proliferation and survival of MSCs, the cells were randomly divided into four groups:YRS, ORS and ORS+DKK1 and ORS+si-β-catenin groups. Changes in cell senescence were tested with SA-β-galactosidase staining and ROS staining. MTT assay was used to test cell proliferation. The survived and apoptotic cells were determined by AO/EB staining after MSCs were treated with H2O2. In order to determine the mechanisms of the Wnt/β-catenin signaling on MSCs aging, immunofluorescence and Western blotting were used to detect the expression of y-H2A.X and p53 protein. RT-PCR were used to detect the expression of p16INK4a, p53 and p21 mRNA. Results Compared with the YRS group, the number of SA-β-galactosidase-positive and ROS-positive cells in the ORS group was significant increased, and the ROS fluorescent level was also clearly increased. MTT assays showed the proliferative capacity of MSCs in the ORS group was significantly reduced than that in the YRS group. AO/EB staining and Hoechest 33342 staining showed that the apoptotic index in the ORS group was significantly increased compared with that in the YRS group, and the MSCs in the ORS group was intolerance for oxidative damage. The Elisa results showed the level of Wnt 3a was increased in ORS than that in YRS. The expression ofβ-catenin in the ORS group was significantly increased and the expression of GSK-3βin the ORS group was remarkable reduced than that in the YRS group. However, after treatmented with DKK1 in ORS, theβ-catenin expression was reduced and the GSK-3βexpression was increased. The results of RT-PCR showed that the expression of c-myc increased in the ORS group than that in the YRS group, and the c-myc expression was ruduced in the ORS+DKK1 group compared with that in the ORS group. The results of RT-PCR, immunofluorescence and Western blotting indicated the second siRNA sequence was the optimal siRNA sequence against P-catenin (si-β-catenin), and the optimal time of transfecting siRNA was 48 h for the si-β-catenin 2. The amount of SA-β-gal positive MSCs in YRS+10 ng/ml Wnt 3a and YRS+50 ng/ml Wnt 3a wasn’t significant increased compared with those cultured in YRS alone, however, in YRS+lOOng/ml Wnt 3 a group, the amount of SA-β-gal positive MSCs was significantly increased. Compared with the ORS group, the number of SA-β-gal positive MSCs those were treated with DKK1 or si-β-catenin was significantly decreased. ROS staining showed the number of ROS-positive cells and the ROS fluorescent level was clearly decreased in the ORS+DKK1 and ORS+ si-β-catenin group than that in the ORS group. After treatment with DKK1 or si-β-catenin to inhibite the Wnt/β-catenin signalling, the proliferative and survival ability of MSCs was significantly increased. The results of immunofluorescence, Western blotting and RT-PCR indicated the expression of y-H2A.X, p16INK4a, p53 and p21 was clearly increased in the ORS group than that in YRS group, however, after treatment with DKK1 or si-β-catenin to inhibite the Wnt/β-catenin signalling, the expression of y-H2A.X, p16INK4a, p53 and p21 was significantly decreased compared with that in the ORS group. Conclusions Our data suggested that ORS can induce the senescence of MSCs and inhibits the proliferation and survival ability of MSCs. The Wnt/p-catenin signaling was clearly activated in the MSCs cultrued with ORS. The activated Wnt/β-catenin signaling can promote MSCs aging. After inhibiting the Wnt/β-catenin signaling, the aging of MSCs was delaied, the proliferative and survival ability of MSCs was increased. The Wnt/β-catenin signaling plays a critical role in MSC aging induced by ORS. The DNA damage response and p53/p21 pathway may be main mediators of MSC aging induced by excessive activation of Wnt/β-catenin signaling. |
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