Papp Tamas, Hollo Krisztina, Meszar-Katona Eva, Nagy Zoltan, Polyak Angela, Miko Edit, Bai Peter, Felszeghy Szabolcs
a Department of Anatomy, Histology and Embryology; Faculty of Medicine , University of Debrecen , Debrecen , Hungary ;
b Department of Medical Chemistry , University of Debrecen , Debrecen , Hungary ;
Acta Odontol Scand. 2016;74(4):307-14. doi: 10.3109/00016357.2015.1130853. Epub 2016 Jan 14.
The aim of this work is to investigate the possible role of Toll-like receptor 4 (TLR4) during the development of mouse tooth germ. TLR4 is well known to inhibit mineralization and cause inflammation in mature odontoblasts and dental pulp cells. However, unlike these pathological functions of TLR4, little is known about the developmental function(s) of TLR4 during tooth development.
TLR4 expression was studied via Western blot in developing lower mouse incisors from E13.5 to E18.5. To generate functional data about the effects of TLR4, a specific agonist (LPS) was applied to the medium of in vitro tooth germ cultures, followed by Western blot, histochemical staining, ELISA assay, in situ hybridization and RT-qPCR.
Increased accumulation of biotin-labelled LPS was detected in the enamel organ and in preodontoblasts. LPS treatment induced degradation of the inhibitor molecule (IκB) of the NF-κB signalling pathway. However, no morphological alterations were detected in cultured tissue after LPS addition at the applied dosage. Activation of TLR4 inhibited the mineralization of enamel and dentin, as demonstrated by alizarin red staining and as decreased levels of collagen type X. mRNA expression of ameloblastin was elevated after LPS administration.
These results demonstrate that TLR4 may decrease the mineralization of hard tissues of the tooth germ and may trigger the maturation of ameloblasts; it can give valuable information to understand better congenital tooth abnormalities.
本研究旨在探讨Toll样受体4（TLR4）在小鼠牙胚发育过程中可能发挥的作用。众所周知，TLR4可抑制成熟成牙本质细胞和牙髓细胞的矿化并引发炎症。然而，与TLR4的这些病理功能不同，其在牙齿发育过程中的发育功能鲜为人知。
通过蛋白质免疫印迹法研究E13.5至E18.5发育阶段的小鼠下切牙中TLR4的表达情况。为了获得有关TLR4作用的功能数据，将一种特异性激动剂（脂多糖，LPS）添加到体外牙胚培养物的培养基中，随后进行蛋白质免疫印迹、组织化学染色、酶联免疫吸附测定、原位杂交和逆转录-定量聚合酶链反应。
在釉质器官和前成牙本质细胞中检测到生物素标记的LPS积累增加。LPS处理诱导核因子κB信号通路的抑制分子（IκB）降解。然而，在所应用的剂量下添加LPS后，培养组织中未检测到形态学改变。茜素红染色和X型胶原水平降低表明，TLR4的激活抑制了釉质和牙本质的矿化。LPS给药后，成釉蛋白的mRNA表达升高。
这些结果表明，TLR4可能会降低牙胚硬组织的矿化，并可能触发成釉细胞的成熟；它可为更好地理解先天性牙齿异常提供有价值的信息。
