The In Vitro Investigation Of LRPPRC Regulating Dental Pulp Stem Cells Senescence

Objective To investigate the phenotypic and molecular changes associated with aging of dental pulp stem cells(DPSCs),and to explore the differential proteins of DPSCs at different ages using proteomic techniques,with a view of providing a theoretical basis for using LRPPRC(Leucine rich pentatricopeptide repeat containing)to delay the aging of DPSCs and promote the clinical translation of DPSCs.Methods DPSCs were isolated and cultured by combined enzymatic digestion and tissue block method,and the morphology of primary and P2 generation DPSCs were observed under microscope.DPSCs were characterized by flow cytometry,osteogenic,lipogenic and chondrogenic induced differentiation.The ageing changes of DPSCs were studied by cell proliferation,migration,senescence-associated β-galactosidase staining and osteogenesis induction assay and detection of senescence-associated gene expression.DPSCs extracted from young(<30y)and old(>40y)individuals were used for further proteomic analysis and screened for differential proteins.Knockdown and overexpression LRPPRC lentiviral vectors were constructed to investigate the effects of LRPPRC overexpression and knockdown lentiviral transfection on senescence of DPSCs.Results Primary DPSCs were microscopically seen to grow radially in the center of the tissue mass,and older P2 generation DPSCs became larger,flattened and longer than the younger DPSCs.DPSCs demonstrated the ability to differentiate into osteogenic,lipogenic,and chondrogenic lineages with their surface marker expression meeting the criteria for identification of mesenchymal stem cells.However,the aged DPSCs showes decreased abilities in proliferation,migration,and osteogenesis,and increased expression of senescence-associated β-galactosidase activity and senescence-associated genes p53 and p16.Proteomics studies showed decreased expression of LRPPRC in aged dental pulp stem cells.Knockdown of LRPPRC resulted in decreased proliferation,migration and osteogenic differentiation ability of dental pulp stem cells,and accelerated senescence with increased expression of senescence-associated β-galactosidase activity and senescence-associated genes p53 and p16.In contrast,overexpression of LRPPRC delayed dental pulp stem cell senescence increased proliferation,migration and osteogenic capacity of DPSCs,and decreased senescence-associated β-galactosidase activity and expression of senescence-associated markers p53 and p16,with statistically significant differences(P < 0.05).Conclusion The present study demonstrates that DPSCs undergo aging with age.LRPPRC plays an important role in delaying the aging of DPSCs and is expected to be an ideal target for action in delaying the aging of DPSCs.Further studies are needed to explore the underlying molecular mechanisms of LRPPRC in DPSC aging and its potential therapeutic applications.