Division of Clinical Cariology and Endodontology, Department of Oral Rehabilitation, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, 061-0293, Japan.
Division of Disease Control and Molecular Epidemiology, Department of Oral Growth and Development, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, 061-0293, Japan.
Odontology. 2021 Jul;109(3):661-671. doi: 10.1007/s10266-020-00588-8. Epub 2021 Jan 21.
The aim of this study was to determine whether histone deacetylase inhibitors (HDACi), including entinostat (MS-275), valproic acid (VPA), trichostatin A (TSA), and sodium butyrate (NaB), promoted the odontogenic differentiation of the odontoblast-like cell line, MDPC-23 in the absence of an osteoblast mineralization medium. The cells were cultured in basal medium (Dulbecco's modified Eagle medium) with and without (controls) the inhibitors. The cell viability and migration were assessed using the cell proliferation reagent WST-1 and a scratch wound healing assay, respectively. The mRNA expression levels of bone morphogenetic protein (Bmp)-2 and -4, collagen 1 alpha 1 (Col1α1), osteocalcin (Oc), dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp), runt-related transcription factor 2 (Runx2), Krueppel-like factor 5 (Klf5), and Msh homeobox 1 (Msx1) were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Alizarin red and alkaline phosphatase assays were performed to determine the extent of mineralization in the culture systems. No significant differences in cell numbers were observed between the controls and the MS-275-, VPA-, and NaB-treated cells; however, a significant difference was observed with TSA (concentration, 1000 nM). The scratch wound healing assay showed no effect of cell migration in the MS-275 (1.0 µM)-treated cells when compared with the controls at 24 h. Furthermore, MS-275, VPA, and NaB increased the mRNA expression levels of Bmp-2 and -4, Oc, and Runx2 followed by the mineralization of the cells. Only MS-275 significantly increased the expression levels of Dmp1, Dspp, Klf5, and Msx1 in the cells. These findings indicated that MS-275 may be considered as a reliable candidate for the odontogenic differentiation of dental pulp cells.
本研究旨在确定组蛋白去乙酰化酶抑制剂(HDACi),包括恩替诺特(MS-275)、丙戊酸(VPA)、曲古抑菌素 A(TSA)和丁酸钠(NaB),是否能在没有成骨细胞矿化培养基的情况下促进成牙本质细胞样细胞系 MDPC-23 的牙源性分化。细胞在基础培养基(改良 Eagle 培养基)中培养,有无抑制剂(对照组)。分别用细胞增殖试剂 WST-1 和划痕愈合试验评估细胞活力和迁移。通过实时定量聚合酶链反应(qRT-PCR)评估骨形态发生蛋白(Bmp)-2 和 -4、胶原 1 阿尔法 1(Col1α1)、骨钙素(Oc)、牙本质基质蛋白 1(Dmp1)、牙本质涎磷蛋白(Dspp)、 runt 相关转录因子 2(Runx2)、 Krueppel 样因子 5(Klf5)和 Msh 同源盒 1(Msx1)的 mRNA 表达水平。用茜素红和碱性磷酸酶试验来确定培养系统中矿化的程度。对照组与 MS-275、VPA 和 NaB 处理的细胞之间细胞数量无显著差异;然而, TSA(浓度为 1000 nM)则有显著差异。划痕愈合试验显示,与对照组相比,MS-275(1.0 μM)处理的细胞在 24 小时时细胞迁移没有影响。此外,MS-275、VPA 和 NaB 增加了 Bmp-2 和 -4、Oc 和 Runx2 的 mRNA 表达水平,随后细胞矿化。只有 MS-275 显著增加了细胞中 Dmp1、Dspp、Klf5 和 Msx1 的表达水平。这些发现表明,MS-275 可被视为牙髓细胞牙源性分化的可靠候选药物。