Department of Pediatric Dentistry, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan.
Department of Biochemistry, Nippon Dental University, School of Life Dentistry at Niigata, Niigata, 1-8, Hamauracho, Chuo-ku, Niigata-Shi, Niigata, 951-8580, Japan.
Arch Oral Biol. 2019 Nov;107:104479. doi: 10.1016/j.archoralbio.2019.07.004. Epub 2019 Jul 8.
Amelogenins are major components of extracellular matrix proteins in developing teeth, and regulate the growth of enamel crystals. They also function as signaling molecules in cell differentiation. This study aimed to determine the biological effects of amelogenins on the differentiation of HAT-7 dental epithelial cells and MC3T3-E1 pre-osteoblastic cells using full-length recombinant human amelogenin (rh-AMEL).
rh-AMEL was expressed in a mammalian cell line (Expi293F™) and was purified by DDK agarose beads. Effects of rh-AMEL on differentiation were evaluated by Mineralization and Alkaline phosphatase (ALP) activity using Alizarin Red S staining and colorimetric substrate p-nitrophenol, respectively.
Western blotting and silver staining confirmed the successful purification of rh-AMEL. Mineralization and ALP activity in HAT-7 cells were significantly higher after treatment with 4 μg/mL rh-AMEL, but not after treatment with Emdogain (EMD). In MC3T3-E1 cells, on the other hand, rh-AMEL showed biphasic effects on differentiation. Treatment with low concentrations of rh-AMEL (0.001-0.1 μg/mL) and EMD (0.01-1 μg/mL) increased mineralization and ALP activity in MC3T3-E1 cells, whereas treatment with high concentrations of rh-AMEL (4 μg/mL) and EMD (100 μg/mL) had the opposite effect.
High concentrations of rh-AMEL and EMD decreased the differentiation of MC3T3-E1 cells. By contrast, a high concentration of rh-AMEL, but not that of EMD, promoted the differentiation of HAT-7 cells. This study demonstrates that the effects of rh-AMEL on cell differentiation differ between HAT-7 and MC3T3-E1 cells, and suggests that different regions on AMEL may induce the differentiation of these cell types.
釉原蛋白是牙发育过程中外细胞基质蛋白的主要成分，调节釉质晶体的生长。它们还作为细胞分化中的信号分子发挥作用。本研究旨在使用全长重组人釉原蛋白（rh-AMEL）确定釉原蛋白对 HAT-7 牙上皮细胞和 MC3T3-E1 前成骨细胞分化的生物学影响。
rh-AMEL 在哺乳动物细胞系（Expi293F™）中表达，并通过 DDK 琼脂糖珠纯化。通过茜素红 S 染色和比色底物对硝基苯酚分别评估 rh-AMEL 对矿化和碱性磷酸酶（ALP）活性的影响。
Western blot 和银染证实 rh-AMEL 的成功纯化。用 4μg/ml rh-AMEL 处理后，HAT-7 细胞的矿化和 ALP 活性显著升高，但用 Emdogain（EMD）处理则没有。另一方面，rh-AMEL 对 MC3T3-E1 细胞的分化表现出双相作用。用低浓度的 rh-AMEL（0.001-0.1μg/ml）和 EMD（0.01-1μg/ml）处理可增加 MC3T3-E1 细胞的矿化和 ALP 活性，而用高浓度的 rh-AMEL（4μg/ml）和 EMD（100μg/ml）处理则有相反的效果。
rh-AMEL 和 EMD 的高浓度降低了 MC3T3-E1 细胞的分化。相比之下，rh-AMEL 的高浓度而非 EMD 的高浓度促进了 HAT-7 细胞的分化。本研究表明，rh-AMEL 对细胞分化的影响在 HAT-7 和 MC3T3-E1 细胞之间存在差异，并提示 AMEL 的不同区域可能诱导这两种细胞类型的分化。
