| Objective:Periodontal ligament stem cells(PDLSCs)have many applications in the field of cytotherapy,tissue engineering,and regenerative medicine.However,the effect of age on the biological and immunological characteristics of PDLSCs remains unclear.Methods:1.Patients were divided into two groups according to their age:the young group(1 9-20 years old)and the elderly group(35-50 years old).The PDLSCs of different age groups were isolated and cultured in vitro by tissue block method.2.To detect the difference of biological characteristics of PDLSCs between the two groups:to compare the expression of surface molecules(STRO-1,CD146,CD31 and CD45)of PDLSCs between the two groups by flow cytometry.CCK-8 and EdU staining were used to compare the proliferation ability of PDLSCs,Annexin V-PE/7-ADD staining was used to compare the apoptosis level of PDLSCs.The two groups of PDLSCs were cultured separately with osteogenic/adipogenic inductive medium,followed by ALP staining and ALP activity seven days later,Alizarin Red staining and quantitative determination 21 days later,and Oil Red O staining and quantitative determination 14 days later,so as to compare the difference of osteogenic and adipogenic differentiation potential of the two groups of PDLSCs.3.To detect the difference of the immunological characteristics of PDLSCs between the two groups:to compare the expression difference of the surface molecules(HLA-?,HLA-? DR,CD80 and CD86)of PDLSCs between the two groups by flow cytometry.Two groups of PDLSCs were co-cultured with allogeneic peripheral blood mononuclear cells(PBMCs),and the proliferation ability of PBMCs co-cultured with two groups of PDLSCs was compared by EdU staining through five different co-cultures.Annexin V-PE/7-ADD staining was used to compare the apoptosis levels of PBMCs co-cultured with PDLSCs of the two groups.4.To explore the underlying mechanism of differences in biological and immunological characteristics of PDLSCs between the two groups:microarray assay was used to compare the differentially expressed genes in PDLSCs between the two groups,and qRT-PCR and western blot analyses were used to verify the highly differentially expressed genes and proteins in PDLSCs between the two groups.Results:1.Two groups of PDLSCs were successfully isolated and cultured,including the young group(YPDLSCs)and the elderly group(APDLSCs).2.The biological characteristics of APDLSCs were significantly weaker than that of YPDLSCs:the positive expression rates of Stro-1 and CD 146 in APDLSCs were lower than those of YPDLSCs.The proliferation capacity of APDLSCs was significantly weaker than that of YPDLSCs.The apoptosis level of APDLSCs was significantly higher than that of YPDLSCs.After 7 days of osteogenic induction,the ALP activity of APDLSCs was significantly lower than that of YPDLSCs.After 21 days of osteogenic induction,the production of mineralized nodules of APDLSCs was significantly lower than that of YPDLSCs.After 14 days of adipogenic induction,the number and size of lipid droplets of APDLSCs were significantly lower than those of YPDLSCs.The osteogenic and adipogenic differentiation potential of APDLSCs were significantly lower than those of YPDLSCs.3.The immunological characteristics of APDLSCs were significantly weaker than that of YPDLSCs:the expression rates of HLA-?,HLA-? DR,CD80 and CD86 showed no statistical difference between APDLSCs and YPDLSCs,and both APDLSCs and YPDLSCs expressed low immunogenicity.Both APDLSCs and YPDLSCs can inhibit the proliferation of allogeneic PBMCs,but the immunosuppressive ability of APDLSCs is weaker than that of YPDLSCs.There was no statistical difference in the apoptosis level of PBMCs co-cultured with APDLSCs and YPDLSCs.4.Compared with YPDLSCs,analysis of mRNA expression revealed an up-regulation of immune related genes(CCND3 and RC3H2),and a down-regulation of osteogenesis related gene(Runx2,ALP,COL1 A1),adipogenic related gene(PPARy2),as well as other immune related genes(CXCL12,FKBPIA,FKBP1B,NCSTN,P2RX7,PPP3CB,RIPK2,SLC11A1,and TP53)in those from APDLSCs.Furthermore,compared with YPDLSCs,the expression levels of osteogenic and adipogenic-related genes and proteins(Runx2,ALP,COL1A1 and PPAR?2)were significantly decreased,while the expression levels of immune-related genes and proteins(CCND3)were significantly increased in APDLSCs.Conclusion:Age influences the biological and immunological characteristics of PDLSCs.The proliferation,differentiation,and immunoregulatory ability of YPDLSCs are better than those of APDLSCs,which means that young PDLSCs should be selected for the treatment of periodontitis or tooth regeneration.Furthermore,we should pay attention to the storage of PDLSCs from young individuals for later use when the donor needs clinical treatment at a later age or for allotransplantation in other elderly people in need. |
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