Zhang Lili, Xia Dengsheng, Wang Chao, Gao Feifei, Hu Lei, Li Juan, Jin Luyuan
Department of General Dentistry and Integrated Emergency Dental Care, Beijing Stomatological Hospital, Capital Medical University, Beijing, China.
Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing, China.
Oral Dis. 2023 Jan;29(1):195-205. doi: 10.1111/odi.13929. Epub 2021 Jun 20.
Pleiotrophin (PTN), a secreted extracellular matrix-associated protein, plays an important role in regulating the osteo/dentinogenic differentiation potential of dental pulp stem cells (DPSCs). Our previous study has demonstrated that PTN expression in young DPSCs was is 10-fold higher than that in aged DPSCs. However, the role of PTN on the in maintaining the stemness of senescent DPSCs remains unclear. The present study aimed to investigate the effect of PTN on senescent DPSCs in vitro.
Dental pulp stem cells were isolated from human third molars. PTN was knocked down using short hairpin RNAs to study the role of PTN on the senescence of DPSCs. DPSCs with aging performance were obtained by a replicative senescence cell model was obtained by the long-term culture of DPSCs to the 15th passage in vitro (P15). We then investigated the effect of PTN on senescent DPSCs (P15 DPSCs). Real-time RT-PCR, western blotting, alizarin red staining, quantitative calcium analysis, SA-β-Gal staining, CFSE, and cell-counting kit-8 (CCK8) assays were used to study cellular senescence and function.
The depletion of PTN increased the ratio of SA-β-gal-positive cells, upregulated the expression of p16, and down-regulated the expression of TERT and p-p38. Furthermore, 50 pg/ml of PTN recombinant protein rescued these changes the altered ratio of SA-β-gal-positive cells, decreased the expression of p16, enhanced TERT and p-p38 expression, as well as telomere activity, caused by PTN depletion and long-term culture. The15th passage cells displayed typical aging characteristic, including high ratio of SA-β-gal-positive cells, increased aging-related gene expression, decreased proliferation rate, high level of Cyclin D expression, and impaired osteo/dentinogenic differentiation potential. However, 50 pg/ml of PTN recombinant protein could partially reverse these alteration rescue these changes.
The present study demonstrated that PTN could protect DPSCs from senescence by improving the proliferation and osteo/dentinogenic differentiation ability, probably through the p38 MAPK pathway.
多效生长因子（PTN）是一种分泌型细胞外基质相关蛋白，在调节牙髓干细胞（DPSCs）的成骨/牙本质分化潜能中起重要作用。我们之前的研究表明，年轻DPSCs中PTN的表达比老年DPSCs高10倍。然而，PTN在维持衰老DPSCs干性方面的作用仍不清楚。本研究旨在探讨PTN对体外衰老DPSCs的影响。
从人类第三磨牙中分离牙髓干细胞。使用短发夹RNA敲低PTN，以研究PTN对DPSCs衰老的作用。通过将DPSCs体外长期培养至第15代（P15）获得具有衰老特征的DPSCs，采用复制性衰老细胞模型。然后我们研究了PTN对衰老DPSCs（P15 DPSCs）的影响。使用实时RT-PCR、蛋白质印迹、茜素红染色、定量钙分析、SA-β-Gal染色、CFSE和细胞计数试剂盒-8（CCK8）检测来研究细胞衰老和功能。
PTN的缺失增加了SA-β-gal阳性细胞的比例，上调了p16的表达，并下调了TERT和p-p38的表达。此外，50 pg/ml的PTN重组蛋白挽救了这些变化，即由PTN缺失和长期培养导致的SA-β-gal阳性细胞比例改变、p16表达降低、TERT和p-p38表达增强以及端粒活性增强。第15代细胞表现出典型的衰老特征，包括SA-β-gal阳性细胞比例高、衰老相关基因表达增加、增殖率降低、细胞周期蛋白D表达水平高以及成骨/牙本质分化潜能受损。然而，50 pg/ml的PTN重组蛋白可以部分逆转这些改变。
本研究表明，PTN可能通过p38 MAPK途径提高增殖和成骨/牙本质分化能力，从而保护DPSCs免于衰老。
