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.
目的： 探讨人牙髓干细胞（hDPSC）随传代次数增加衰老相关表型和分子的改变，探究含WW域转录调节因子1（WWTR1）在hDPSC衰老中的作用。 方法： 组织块法原代培养hDPSC，根据培养出的hDPSC来源受试者的年龄、细胞代数及细胞敲低和过表达WWTR1后将细胞分为4组。Ⅰ组：人牙来源的hDPSC根据受试者不同年龄分为青年组（15~25岁）和中年组（40~50岁）。Ⅱ组：根据hDPSC不同传代分为年轻细胞组（传至第3代）和年老细胞组（传至第10代）。Ⅲ组：hDPSC敲低WWTR1，分为敲低组和敲低空载体组。Ⅳ组：hDPSC过表达WWTR1，分为过表达组和过表达空载体组。实时荧光定量PCR（RT-qPCR）检测Ⅰ、Ⅱ组WWTR1表达量的变化，对Ⅱ、Ⅲ、Ⅳ组通过细胞计数试剂盒（CCK-8）检测细胞增殖能力、茜素红染色检测细胞成骨分化能力、衰老相关β-半乳糖苷酶染色检测细胞衰老阳性率、RT-qPCR检测衰老相关基因p53、p21表达量的变化。 结果： 衰老细胞占比随连续传代培养逐渐增加，与年轻细胞组相比，年老细胞组hDPSC的增殖及成骨分化能力均显著降低（P<0.001），年老细胞组hDPSC衰老相关基因p53（2.09±0.24）、p21（4.91±0.54）的表达量均显著高于年轻细胞组［p53：（1.08±0.09），p21：（1.09±0.08）］（P<0.01，P<0.001）。与青年组和年轻细胞组hDPSC相比，中年组和年老细胞组hDPSC的WWTR1表达量均显著降低（P<0.01）。敲低组（44.50±2.42）较敲低空载体组（22.27±0.56）衰老细胞占比显著增加（P<0.001），hDPSC敲低WWTR1后衰老相关基因p53、p21的表达水平显著上调（P<0.001），敲低组hDPSC的增殖及成骨分化能力较敲低空载体组显著降低（P<0.001）。过表达空载体组（20.40±0.79）较过表达组（10.07±0.61）衰老细胞占比显著增加（P<0.001），hDPSC过表达WWTR1后衰老相关基因p53、p21的表达水平显著下调，过表达组hDPSC的增殖及成骨分化能力较过表达空载体组显著升高（P<0.001）。 结论： WWTR1可以抑制衰老相关基因p53、p21的表达进而延缓hDPSC衰老，且能提高hDPSC的增殖和成骨分化能力。.