低浓度氟化钠对人牙髓细胞的成骨/成牙本质分化的影响

Effect of low concentration of sodium fluoride on osteogenic/odontogenic differentiation of human dental pulp cells

摘要

目的 探讨低浓度氟化钠对人牙髓细胞（human dental pulp cells，hDPCs）成骨/成牙本质分化的影响。方法 本研究已通过单位伦理委员会审查批准。原代培养hDPCs，采用MTT法检测不同浓度氟化钠对hDPCs增殖的影响；选取合适浓度的氟化钠加入成骨/成牙本质分化诱导培养液中，对hDPCs进行体外诱导，通过茜素红染色检测hDPCs成骨/成牙本质分化能力的变化，RT-qPCR检测分化相关基因的mRNA表达；同时通过RT-qPCR和Western blot检测hDPCs成骨/成牙本质分化过程中内质网应激相关基因的表达。结果 低浓度氟化钠（0.1 mmol/L）在体外可刺激hDPCs增殖，高浓度氟化钠（5 ~ 10 mmol/L）可抑制hDPCs增殖（P<0.05）。选取0.1 mmol/L氟化钠体外混合成骨/成牙本质分化诱导培养后hDPCs的茜素红染色增加，成骨/成牙本质分化相关基因牙本质涎磷蛋白（dentin sialophosphoprotein，DSPP）、骨涎蛋白（bone sialoprotein，BSP）和骨钙蛋白（osteocalcin，OCN）mRNA表达水平升高（P<0.05）。同时在此过程中RT-qPCR检测出mRNA水平hDPCs内质网应激相关基因：剪切x盒结合蛋白1（splicing x-box binding protein-1，sXBP1）、葡萄糖调节蛋白78（glucose-regulated protein 78，GRP78）以及活化转录因子4（activating transcription factor 4，ATF4）表达升高（P<0.05）；Western blot检测出氟化钠混合成骨/成牙本质分化培养后细胞磷酸化真核起始因子-2α（phosphorylated eukaryotic initiation factor-2α，p-eIF2α）、磷酸化蛋白激酶样内质网激酶（phosphorylated the RNA-activated protein kinase-like ER-resident kinase，p-PERK）和ATF4蛋白表达增加（P<0.05）。结论 低剂量氟化钠促进人牙髓细胞的成骨/成牙本质分化并伴有内质网应激水平的升高。

Abstract

Objective To study the effect of low concentrations of sodium fluoride on the osteogenic/odontogenic differentiation of human dental pulp cells (hDPCs) in vitro. Methods This study was reviewed and approved by the Ethics Committee. hDPCs were cultured using a modified tissue explant technique in vitro. The effects of different concentrations of sodium fluoride on the proliferation of hDPCs were measured by methylthiazol tetrazolium (MTT) assay. Appropriate concentrations were added to the osteogenic/odontogenic differentiation induction medium, and the cells were induced in vitro. Alizarin red S staining was used to detect the osteoblastic/odontogenic differentiation ability of the cells, and the mRNA expression of the key differentiation factors was detected by RT-qPCR. Moreover, the expression of key molecules of endoplasmic reticulum stress (ERS) was detected by RT-qPCR and Western blot. The data were analyzed with the SPSS 18.0 software package. Results Low concentration of NaF (0.1 mmol/L) could stimulate cell proliferation in vitro, while a high concentration (5-10 mmol/L) could inhibit cell proliferation (P<0.05). According to the literature and the experimental data, 0.1 mmol/L NaF was selected as the following experimental concentration. The levels of alizarin red S staining were increased after NaF induction of mixed osteogenic/odontogenic differentiation in vitro. The mRNA expression levels of key molecules for osteogenic/odontogenic differentiation, dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP) and osteocalcin (OCN), were increased (P<0.05). The mRNA levels of ERS markers (splicing x-box binding protein-1 (sXBP1), glucose-regulated protein 78 (GRP78) and activating transcription Factor 4 (ATF4) were increased in NaF-treated cells. The protein expression levels of key ER stress molecules (phosphorylated RNA-activated protein kinase-like ER-resident kinase (p-PERK), phosphorylated eukaryotic initiation factor-2α (p-eIF2α) and ATF4) were higher in NaF-treated cells. Conclusion A low concentration of NaF promotes the osteogenic/odontogenic differentiation of hDPCs and increases the level of ER stress.

关键词

人牙髓细胞 / 氟化钠 / 增殖 / 成骨/成牙本质分化 / 内质网应激 / 剪切X盒结合蛋白1 / 活化转录因子4 / 葡萄糖调节蛋白78 / 蛋白激酶样内质网激酶 / 真核起始因子-2α

Key words

human dental pulp cell / sodium fluoride / proliferation / osteogenic/odontogenic differentiation / endoplasmic reticulum stress / splicing xbox binding protein 1 / activating transcription factor 4 / glucose-regulated protein 78 / RNA-activated protein kinase-like ER-resident kinase / eukaryotic initiation factor-2α

Author summay

李莉芬，主治医师，博士，Email：[e-mail]