Awada Tetsuya, Kunimatsu Ryo, Yoshimi Yuki, Hirose Naoto, Mitsuyoshi Tomomi, Sumi Keisuke, Tanimoto Kotaro
Department of Orthodontics, Applied Life Sciences, Hiroshima University, Institute of Biomedical & Health Sciences, Japan.
Department of Orthodontics, Applied Life Sciences, Hiroshima University, Institute of Biomedical & Health Sciences, Japan.
Biochem Biophys Res Commun. 2017 Jan 22;482(4):1154-1159. doi: 10.1016/j.bbrc.2016.12.003. Epub 2016 Dec 2.
Amelogenins, enamel matrix proteins secreted by ameloblasts, comprise 90% of the developing extracellular enamel matrix. Recent evidence suggests that amelogenins might induce the proliferation of various cells. However, the residues comprising the active site of amelogenin remain unclear. Therefore, this study aimed to examine the effects of a human amelogenin C-terminal peptide (amgCP) on the metabolism of osteoblasts.
Mouse calvarial osteoblastic cells (MC3T3-E1) were cultured and treated with amgCP. Cell proliferation was measured using MTS and BrdU assays. After confluence was reached, the cells were cultured in osteogenic differentiation medium and treated with 0, 100, or 1000 ng/ml amgCP. Cell differentiation activity was examined by real-time PCR, western blotting, and ALP activity. Mineralization was evaluated by Alizarin red staining.
Cell numbers of MC3T3-E1 were significantly (P < 0.05) increased by treatment with 1000 ng/ml amgCP as compared to the control group at 4 and 6 days. In addition, the proliferative activity of MC3T3-E1 was significantly enhanced by treatment with 100 or 1000 ng/ml amgCP. The mRNA levels and protein expressions of ALP and BSP were not changed by treatment with amgCP as compared to the non-treated controls on days 7 and 14. The osteogenic differentiation of MC3T3-E1 cells was not affected by treatment with amgCP as compared with untreated controls.
The C-terminus of amelogenin promotes the proliferation of MC3T3-E1 cells, indicating the possible utility of the C11 peptide in bone-tissue regeneration.
釉原蛋白是成釉细胞分泌的釉质基质蛋白，占发育中的细胞外釉质基质的90%。最近的证据表明，釉原蛋白可能诱导多种细胞的增殖。然而，构成釉原蛋白活性位点的残基仍不清楚。因此，本研究旨在探讨人釉原蛋白C末端肽（amgCP）对成骨细胞代谢的影响。
培养小鼠颅骨成骨细胞（MC3T3-E1）并用amgCP处理。使用MTS和BrdU测定法测量细胞增殖。达到汇合后，将细胞在成骨分化培养基中培养并用0、100或1000 ng/ml amgCP处理。通过实时PCR、蛋白质印迹和碱性磷酸酶（ALP）活性检测细胞分化活性。用茜素红染色评估矿化情况。
与对照组相比，在第4天和第6天用1000 ng/ml amgCP处理后，MC3T3-E1的细胞数量显著增加（P < 0.05）。此外，用100或1000 ng/ml amgCP处理可显著增强MC3T3-E1的增殖活性。与未处理的对照组相比，在第7天和第14天用amgCP处理后，ALP和骨涎蛋白（BSP）的mRNA水平和蛋白质表达没有变化。与未处理的对照组相比，amgCP处理对MC3T3-E1细胞的成骨分化没有影响。
釉原蛋白的C末端促进MC3T3-E1细胞的增殖，表明C11肽在骨组织再生中可能具有应用价值。
