钙通过调节氟诱导的内质网应激反应上调成釉器细胞中KLK4和AMTN表达

The biological mechanism of calcium nutrition deducing dental fluorosis is still not clear. We found that calcium could up-regulate the KLK4 and AMTN expression of enamel organ cells. Moreover, KLK4 and AMTN mRNA expression of enamel organ cells increased and the phosphorylation PERK deduced. Basing on our previous work and the above results, we propose that calcium may play its role in dental fluorosis by raising KLK4 and AMTN expression. In this study, the influence of calcium on dental fluorosis is observed by animal model, and the biochemical indicators, clinical phenotype and ultrastructure of mandibular incisors were observed to identify the antagonistic effect of calcium in dental fluorosis; That the influence of calcium on regulating the KLK4 and AMTN mRNA expression of enamel organ cells exposed to fluoride is observed via animal and cell experiment by RT-PCR and Western-blot. At the same time, that calcium regulates ER stress through PERK/eIF2α signal way is study to further explore the cell biological mechanism that calcium effects the expression of KLK4 and AMTN. The results of this study will provide a theoretical basis for the application of calcium in dental fluorosis prevention and reasonable application of fluoride.

钙拮抗氟的牙毒性的细胞分子学机制目前尚不明确。我们发现钙可直接上调成釉器细胞中KLK4和AMTN的表达，且调节氟对KLK4和AMTN mRNA表达及PERK磷酸化的影响。结合前期工作提出钙可通过调节氟诱导的PERK/eIF2α介导的内质网应激反应以上调KLK4和AMTN表达，从而拮抗氟牙症的发生。本研究将通过钙干预氟牙症大鼠，对其下颌切牙表型进行临床及超微结构观察，确立钙对氟的牙毒性的拮抗作用；通过钙干预动物及体外细胞，利用实时定量RT-PCR和Western-blot等分子生物学技术，观察钙对氟下调成釉器细胞中KLK4和AMTN表达的调节作用及对内质网应激反应的调节作用，探讨钙通过调节PERK/eIF2α介导的内质网应激反应影响KLK4和AMTN表达从而拮抗氟牙症发生的细胞学机制，以增加钙拮抗氟牙症的细胞分子水平的认识，促进补钙在氟牙症的防治中的应用及合理应用氟化物。

氟牙症是牙齿在发育过程中机体摄入过量的氟导致的牙釉质矿化和发育异 常，是地方性氟中毒最早的特异性体征。已有研究表明过量氟可通过激活内质网应激反应下调釉基质蛋白酶激肽释放酶4（KLK4）和釉成熟蛋白（AMTN）的表达，而且补钙可能是预防氟牙症、提高硬组织矿化程度的有效手段，但补钙抗氟牙症损害的机制目前尚不完全明确。本项目通过动物实验发现补充钙营养可减轻氟牙症临床症状；通过动物及细胞实验，利用Western-blot等分子生物学技术，发现补钙对过量氟导致的KLK4表达下调具有调节作用，并发现补钙对过量氟引起的网应激反应蛋白GRP78,PERK 和 eIF2α磷酸化及 AFT4、CHOP表达下调具有抑制作用，说明钙营养可通过抑制内质网应激反应调节KLK4的表达，并减少成釉器细胞的凋亡，从而拮抗氟牙症的发生。该结果有助于补钙在氟牙症的防治中的应用，并为合理应用氟化物增加了理论基础和依据。