Department of Oral and Maxillofacial Pathobiology, Hiroshima University, Hiroshima 734-8553, Japan.
Arch Oral Biol. 2011 Apr;56(4):374-9. doi: 10.1016/j.archoralbio.2010.10.012. Epub 2010 Nov 11.
This study investigates the effect of the N-terminal region of a synthetic porcine ameloblastin peptide on the proliferation and differentiation of human periodontal ligament cells (PDLC).
We used a cell counter to assess the effect of ameloblastin peptides on the proliferation of PDLC. To investigate the effect of ameloblastin peptides on the differentiation of PDLC, we examined quantitative analysis of alkaline phosphatase (ALP) activity by the Bessey-Lowry enzymological method, mineral nodule formation by Dahl's method, and expression of mineralization-related genes by RT-PCR. We used an anti-ameloblastin antibody to determine whether stimulation of ALP activity was caused by the peptide.
At all concentrations examined, the effect of the ameloblastin peptide on cell proliferation was not significantly different compared with the control. However, the peptide significantly stimulated ALP activity in a dose-dependent manner. ALP activity was significantly inhibited by an anti-ameloblastin antibody, which caused ALP levels to revert to their approximate levels in the untreated condition. At concentrations greater than 1ng/ml, the peptide promoted mineralized nodule formation of PDLC. And the peptide induced higher expressions of ALP and bone sialoprotein (BSP) than the control.
Our results show that the ameloblastin peptide upregulate ALP and BSP levels and can enhance calcification of PDLC. Thus, we suggest that the N-terminal synthetic ameloblastin peptide promotes the differentiation activity of PDLC.
本研究旨在探讨合成猪釉原蛋白肽的 N 端区域对人牙周膜细胞（PDLC）增殖和分化的影响。
我们使用细胞计数器评估釉原蛋白肽对 PDLC 增殖的影响。为了研究釉原蛋白肽对 PDLC 分化的影响，我们通过贝塞斯-洛里酶学法对碱性磷酸酶（ALP）活性进行定量分析，通过达尔法检测矿化结节的形成，通过 RT-PCR 检测矿化相关基因的表达。我们使用抗釉原蛋白抗体来确定 ALP 活性的刺激是否由肽引起。
在所有检查的浓度下，与对照组相比，釉原蛋白肽对细胞增殖的影响没有显著差异。然而，该肽以剂量依赖的方式显著刺激 ALP 活性。ALP 活性被抗釉原蛋白抗体显著抑制，使 ALP 水平恢复到未处理状态下的近似水平。在浓度大于 1ng/ml 时，该肽促进 PDLC 的矿化结节形成。并且，该肽诱导的 ALP 和骨涎蛋白（BSP）表达水平高于对照组。
我们的结果表明，釉原蛋白肽上调 ALP 和 BSP 水平，并能增强 PDLC 的钙化。因此，我们认为合成釉原蛋白肽的 N 端促进 PDLC 的分化活性。
