Establishment of an aging model of mesenchymal stem cells / 军事医学

ABSTRACT

Objective To establish an aging model of mesenchymal stem cells (MSCs) and to investigate aging related biological mechanism for the purpose of studying the senesence of MSCs .Methods MSCs were separated and purified from human placenta, and the cells of the third passage(P3-MSCs) were cultured in the medium for 2 hours, then 100,200 and 300 μmol/L hydrogen peroxide ( H2 O2 ) was added to the cells for 2 hours to establish the MSCs aging model in vitro. Biological characteristics of aging MSCs were evaluated by cell cycle assay and senescence associated β-galactosidase staining.The expression of p16,p21 and p53 genes was further measured using quantitative real-time PCR (RT-PCR).Re-sults Compared with the control , the number of MSCs treated with 200μmol/L H2 O2 for 2 hours was significantly decreased and the cells displayed less adipogenic ,osteogenic and chondrogenic differentiation .Moreover ,after exposure to 200 μmol/L H2 O2 , the majority of the cells were in the G 0/G1 phase as showed by cell cycle analysis .The percentage of senescence-associated β-galactosidase-positive cells was increased , and the expression of p 16 , p21 and p53 mRNA and protein was significantly increased.Conclusion The results of this study has demonstrated that the H 2 O2 (200 μmol/L) can be used to establish the aging model of MSCs in vitro, and the cellular phenotypic alteration may attribute to the cell cycle associated gene expression (p16, p21, and p53).