Li D D, Liu H J, Wang Y, Chen Z G, Zhang X, Li W J
Department of Stomatology, The Second Hospital of Hebei Medical University, Shijiazhuang050000, China.
Department of Key Laboratory, School and Hospital of Stomatology, Hebei Medical University & Hebei Key Laboratory of Stomatology & Hebei Clinical Research Center for Oral Diseases, Shijiazhuang050017, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2024 Dec 9;59(12):1240-1247. doi: 10.3760/cma.j.cn112144-20240521-00212.
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.
随着人牙髓干细胞（hDPSC）传代次数增加，研究与衰老相关的表型和分子变化，以探讨含WW转录调节因子1（WWTR1）在衰老机制中的作用。采用组织块法培养hDPSCs，并根据hDPSCs的年龄、代数、细胞敲低和过表达WWTR1分为4组。Ⅰ组：人牙来源的hDPSCs根据不同年龄进一步分为青年组（15 - 25岁）和中年组（40 - 50岁）。Ⅱ组：根据传代不同，hDPSCs分为年轻细胞组（hDPSCs传至P3代）和老年细胞组（hDPSCs传至P10代）。Ⅲ组：hDPSCs敲低WWTR1，进一步分为敲低组和敲低载体组。Ⅳ组：hDPSCs过表达WWTR1，进一步分为过表达组和过表达载体组。采用实时荧光定量PCR（RT-qPCR）检测Ⅰ组和Ⅱ组中WWTR1表达变化，Ⅱ、Ⅲ、Ⅳ组采用细胞计数试剂盒-8（CCK-8）法。通过CCK-8法检测细胞增殖能力。采用茜素红染色检测成骨分化能力。采用衰老相关β-半乳糖苷酶染色检测细胞衰老阳性率。采用RT-qPCR检测衰老相关基因p53和p21的表达水平。随着连续培养，衰老细胞比例逐渐增加。老年组hDPSCs的增殖和成骨分化能力明显低于青年组（<0.001）。老年细胞组中衰老相关基因p53（2.09±0.24）和p21（4.91±0.54）的表达水平分别高于年轻细胞组[p53：（1.08±0.09）和p21：（1.09±0.08）]（<0.01，<0.001）。中年组和老年细胞组hDPSCs的WWTR1表达水平均低于青年组和年轻细胞组（<0.01）。敲低组衰老细胞比例（44.50±2.42）高于敲低载体组（22.27±0.56）（<0.001）。hDPSCs敲低WWTR1后，衰老相关基因p53和p21的表达水平上调（<0.001），敲低组的增殖和成骨分化能力低于敲低载体组（<0.001）。过表达空载体组衰老细胞比例（20.40±0.79）高于过表达组（10.07±0.61）（<0.001）。hDPSCs过表达WWTR1后，衰老相关基因p53和p21的表达水平下调，过表达组的增殖和成骨分化能力高于过表达载体组（<0.001）。WWTR1可抑制衰老相关基因p53和p21的表达水平，从而延缓衰老进程，并促进hDPSCs的增殖和成骨分化。
