Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou, China.
College of Stomatology, Southern Medical University, Guangzhou, China.
J Cell Mol Med. 2018 Jul;22(7):3442-3451. doi: 10.1111/jcmm.13621. Epub 2018 Apr 14.
The mineralization of dental pulp stem cells is an important factor in the tissue engineering of teeth, but the mechanism is not yet obvious. This study aimed to identify the effect of Stathmin on the proliferation and osteogenic/odontoblastic differentiation of human dental pulp stem cells (hDPSCs) and to explore whether the Shh signalling pathway was involved in this regulation. First, Stathmin was expressed in the cytoplasm and on the cell membranes of hDPSCs by cell immunofluorescence. Then, by constructing a lentiviral vector, the expression of Stathmin in hDPSCs was inhibited. Treatment with Stathmin shRNA (shRNA-Stathmin group) inhibited the ability of hDPSCs to proliferate, as demonstrated by a CCK8 assay and flow cytometry analysis, and suppressed the osteogenic/odontoblastic differentiation ability, as demonstrated by alizarin red S staining and osteogenic/odontoblastic differentiation-related gene (ALP, BSP, OCN, DSPP) activity, compared to that of hDPSCs from the control shRNA group. Molecular analyses showed that the Shh/GLI1 signalling pathway was inhibited when Stathmin was silenced, and purmorphamine, the Shh signalling pathway activator, was added to hDPSCs in the shRNA-Stathmin group, real-time PCR and Western blotting confirmed that expression of Shh and its downstream signalling molecules PTCH1, SMO and GLI1 increased significantly. After activating the Shh signalling pathway, the proliferation of hDPSCs increased markedly, as demonstrated by a CCK8 assay and flow cytometry analysis; osteogenic/odontoblastic differentiation-related gene (ALP, BSP, OCN, DSPP) expression also increased significantly. Collectively, these findings firstly revealed that Stathmin-Shh/GLI1 signalling pathway plays a positive role in hDPSC proliferation and osteogenic/odontoblastic differentiation.
牙髓干细胞的矿化是牙齿组织工程的一个重要因素，但机制尚不清楚。本研究旨在鉴定 Stathmin 对人牙髓干细胞（hDPSCs）增殖和成骨/成牙本质分化的影响，并探讨 Shh 信号通路是否参与这种调节。首先，通过细胞免疫荧光法，Stathmin 在 hDPSCs 的细胞质和细胞膜上表达。然后，通过构建慢病毒载体，抑制 hDPSCs 中 Stathmin 的表达。Stathmin shRNA（shRNA-Stathmin 组）处理抑制了 hDPSCs 的增殖能力，这通过 CCK8 检测和流式细胞术分析得到证实，并且抑制了成骨/成牙本质分化能力，这通过茜素红 S 染色和骨/牙本质分化相关基因（ALP、BSP、OCN、DSPP）活性得到证实，与对照组 hDPSCs 相比。分子分析表明，沉默 Stathmin 时抑制了 Shh/GLI1 信号通路，并且在 shRNA-Stathmin 组中添加 Shh 信号通路激活剂 purmorphamine，实时 PCR 和 Western blot 证实 Shh 及其下游信号分子 PTCH1、SMO 和 GLI1 的表达显著增加。激活 Shh 信号通路后，hDPSCs 的增殖明显增加，这通过 CCK8 检测和流式细胞术分析得到证实；成骨/牙本质分化相关基因（ALP、BSP、OCN、DSPP）的表达也显著增加。总之，这些发现首次表明 Stathmin-Shh/GLI1 信号通路在 hDPSC 增殖和成骨/成牙本质分化中发挥积极作用。
