[Effect of WW-domain transcription regulator 1 on aging regulation of human dental pulp stem cells].

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. 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. 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 (<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)] (<0.01, <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 (<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) (<0.001). After knocking down WWTR1 in hDPSCs, the expression levels of age-related genes p53 and p21 were up-regulated (<0.001), and the abilities of proliferation and osteogenic differentiation in the knockdown group were lower than those in the knockdown carrier group (<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) (<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 (<0.001). 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.