Objective Investigating the changes of phenotype and moleculars associated with aging with the increase of passage times of human dental pulp stem cells(hDPSC),to explore the role of WW-containing transcriptional regulator 1(WWTR1)in the aging mechanism.Methods hDPSCs were cultured by tissue block method,and were divided into 4 groups according to the age,algebra,cell knockdown and overexpression of WWTR1 in hDPSCs.Group Ⅰ:hDPSCs from human teeth were further divided into youth group(15-25 years old)and group middle-aged group(40-50 years old)according to different ages.Group Ⅱ:according to different passage,hDPSCs were divided into young cells group(hDPSCs were transmitted to P3 generation),and old cells group(hDPSCs were transmitted to P10 generation).Group Ⅲ:hDPSCs were knocked down of WWTR1,which were further divided into knockdown group and knockdown carrier group.Group IV:hDPSCs were overexpressed of WWTR1,which were further divided into overexpression group and overexpression carrier group.Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the changes of WWTR1 expression in groups Ⅰ and Ⅱ,and cell counting kit-8(CCK-8)was used for groups Ⅱ,Ⅲ,and Ⅳ.Cell proliferation capacity was detected by CCK-8 assay.The ability of osteogenic differentiation was detected by alizarin red staining.Cell senescence positive rate was detected by age-related β-galactosidase staining.The expression levels of age-related genes p53 and p21 were detected by RT-qPCR.Results The proportion of senescent cells increased gradually with continuous culture.The proliferation and osteogenic differentiation of hDPSCs in the old group were significantly lower than those in the young group(P＜0.001).The expression levels of senescence related genes p53(2.09±0.24)and p21(4.91±0.54)in old cell group were higher than those in young cell group respectively[p53:(1.08±0.09)and p21:(1.09±0.08)](P＜0.01,P＜0.001).The WWTR1 expression levels of hDPSCs in middle-aged group and old cells group were both decreased compared with those in young group and young cells group(P＜0.01).The proportion of senescent cells in knockdown group(44.50±2.42)was higher than that in knockdown carrier group(22.27±0.56)(P＜0.001).After knocking down WWTR1 in hDPSCs,the expression levels of age-related genes p53 and p21 were up-regulated(P＜0.001),and the abilities of proliferation and osteogenic differentiation in the knockdown group were lower than those in the knockdown carrier group(P＜0.001).The proportion of senescent cells in overexpression empty carrier group(20.40±0.79)was higher than that in overexpression group(10.07±0.61)(P＜0.001).After WWTR1 overexpression ins hDPSCs,the expression levels of age-related genes p53 and p21 were down-regulated,and the proliferation and osteogenic differentiation ability in overexpression group were higher than those in overexpression carrier group(P＜0.001).Conclusions WWTR1 can inhibit the expression levels of age-related genes p53 and p21,thus delaying the aging process as well as promoting the proliferation and osteogenic differentiation of hDPSCs.
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