ABSTRACT

Purpose: Pleiotrophin (PTN) is a heparin-binding growth-associated molecule and expressed in ameloblasts and odontoblasts throughout tooth maturation. Our previous study has shown that PTN expressed more than 20-fold higher in dental tissue than dental stem cells. However, the role of PTN on proliferation and osteo/dentinogenesis of dental pulp stem cells (DPSCs) is unclear. The purpose of the present study was to investigate the role of PTN on the DPSCs’ function.

Methods: DPSCs were isolated from human third molars. Short hairpin RNAs (shRNAs) was used to knock down the PTN expression in DPSCs. Real-time RT-PCR, alizarin red staining, quantitative calcium analysis, in vivo transplantation and cell counting kit-8 (CCK8) assay were used to study the function of DPSCs. Possible mechanism was studied by RNA sequencing.

Results: After PTN depletion, ALP activity and mineralization ability of DPSCs decreased. Expression of DMP-1 and BSP weakened. Proliferation of DPSCs at 48 h and 72 h was inhibited. Furthermore, 50 pg/mL PTN recombinant protein rescued the impaired osteo/dentinogenic differentiation potential and proliferation ability caused by PTN depletion. In addition, RNA sequencing showed 221 genes were downregulated and 233 genes upregulated in PTN depleted DPSCs. Several genes including BMP2 and IGFBP5 might be associated with PTN function on the DPSCs. P53 and the AMPK signaling pathways were involved. LncRNA analysis displayed 47 significantly upregulated lncRNA and 31 downregulated lncRNA comparing PTN depleted DPSCs with the control.

Conclusion: Our research demonstrated that PTN has a positive role in maintaining DPSCs proliferation and osteo/dentinogenic differentiation potential.

摘要

目的：多效素 (PTN) 是一种肝素结合生长相关分子，在整个牙齿成熟过程中在成釉细胞和成牙本质细胞中表达。我们之前的研究表明，PTN 在牙齿组织中的表达比牙齿干细胞高 20 倍以上。然而，PTN对牙髓干细胞（DPSCs）增殖和骨/牙本质形成的作用尚不清楚。本研究的目的是调查 PTN 对 DPSCs 功能的作用。

方法：从人类第三磨牙中分离出DPSCs。短发夹 RNA (shRNA) 用于敲低 DPSCs 中的 PTN 表达。实时RT-PCR、茜素红染色、定量钙分析、体内移植和细胞计数试剂盒8（CCK8）测定用于研究DPSCs的功能。通过RNA测序研究了可能的机制。

结果： PTN耗竭后，DPSCs的ALP活性和矿化能力下降。DMP-1和BSP的表达减弱。DPSCs 在 48 小时和 72 小时的增殖受到抑制。此外，50 pg/mL PTN 重组蛋白挽救了由 PTN 耗竭引起的骨/牙本质分化潜能和增殖能力受损。此外，RNA 测序显示 PTN 耗尽的 DPSCs 中有 221 个基因被下调，233 个基因被上调。包括 BMP2 和 IGFBP5 在内的几个基因可能与 DPSCs 上的 PTN 功能有关。涉及 P53 和 AMPK 信号通路。LncRNA 分析显示 47 个显着上调的 lncRNA 和 31 个下调的 lncRNA，将 PTN 耗尽的 DPSC 与对照进行比较。

结论：我们的研究表明，PTN 在维持 DPSCs 增殖和成骨/牙本质分化潜能方面具有积极作用。