Zhang Ying, Zhang KaiQiang, Ma Lin, Gu HeFeng, Li Jian, Lei Shuang
Shanghai Stomatology Hospital, Department of Preventive Dentistry, Fudan University, Shanghai, China.
Department of Preventive Dentistry, School of Stomatology, China Medical University, Shenyang, Liaoning, China; Liaoning Institute of Dental Research, China.
Arch Oral Biol. 2016 Sep;69:95-101. doi: 10.1016/j.archoralbio.2016.05.015. Epub 2016 May 26.
The aim of the study was to evaluate the involvement of endoplasmic reticulum stress and intracellular calcium overload on the development of dental fluorosis.
We cultured and exposed rat ameloblast HAT-7 cells to various concentrations of fluoride and measured apoptosis with flow cytometry and intracellular Ca2+ changes using confocal microscopy, investigated the protein levels of GRP78, calreticulin, XBP1 and CHOP by western blotting, and their transcriptional levels with RT-PCR. We also created an in vivo model of dental fluorosis by exposing animals to various concentrations of fluoride. Subsequently, thin dental tissue slices were analyzed with H&E staining, immunohistochemical staining, and transmission electron microscopy, TUNEL assay was also performed on dental tissue slices for assessment of apoptosis.
High fluoride concentration was associated with decreased ameloblast proliferation, elevated ameloblast apoptosis, and increased intracellular Ca2+ in vitro. The translation and transcription of the proteins associated with endoplasmic reticulum stress were significantly elevated with high concentrations of fluoride. Based on immunohistochemical staining, these proteins were also highly expressed in animals exposed to high fluoride concentrations. Histologically, we found significant fluorosis-like changes in tissues from animals exposed to high fluoride concentrations. Transmission electron microscopy cytology indicated significant apoptotic changes in tissues exposed to high concentrations of fluoride.
These results indicate that exposure to high levels of fluoride led to endoplasmic reticulum stress which induced apoptosis in cultured ameloblasts and in vivo rat model, suggesting an important role of calcium overload and endoplasmic reticulum stress triggered by high concentrations of fluoride in the development of dental fluorosis.
本研究旨在评估内质网应激和细胞内钙超载在氟斑牙发生发展中的作用。
我们培养大鼠成釉细胞HAT-7细胞，并将其暴露于不同浓度的氟化物中，通过流式细胞术检测细胞凋亡情况，利用共聚焦显微镜测量细胞内Ca2+的变化，通过蛋白质印迹法研究葡萄糖调节蛋白78（GRP78）、钙网蛋白、X盒结合蛋白1（XBP1）和C/EBP同源蛋白（CHOP）的蛋白水平，并用逆转录聚合酶链反应（RT-PCR）检测其转录水平。我们还通过让动物暴露于不同浓度的氟化物中建立了氟斑牙的体内模型。随后，对薄的牙齿组织切片进行苏木精-伊红（H&E）染色、免疫组织化学染色和透射电子显微镜检查，还对牙齿组织切片进行TUNEL检测以评估细胞凋亡情况。
在体外，高氟浓度与成釉细胞增殖减少、成釉细胞凋亡增加以及细胞内Ca2+升高有关。高浓度氟化物使与内质网应激相关的蛋白质的翻译和转录显著升高。基于免疫组织化学染色，这些蛋白质在暴露于高氟浓度的动物中也高度表达。组织学上，我们在暴露于高氟浓度的动物组织中发现了明显的氟斑牙样变化。透射电子显微镜细胞学检查表明，暴露于高浓度氟化物的组织中有明显的凋亡变化。
这些结果表明，暴露于高水平氟化物会导致内质网应激，进而诱导培养的成釉细胞和体内大鼠模型发生凋亡，提示高浓度氟化物引发的钙超载和内质网应激在氟斑牙的发生发展中起重要作用。
