A search for the mechanism of the stretch-induced activation of human periodontal ligament cells

本研究旨在阐明人牙周膜细胞对周期性牵张的反应。我们专注于拉伸的hPDL细胞的破骨细胞的分化和血管内皮生长因子在拉伸的hPDL细胞的生产的作用。结果如下：1）RT-PCR和北方印迹法显示，在含10%FCS的αMEM培养基中，在1 α，25（OH）_2维生素D_3存在下，培养的hPDL细胞表达NF-κ B B配体受体激活因子（RANKL）及其诱饵受体骨保护素（OPG）的mRNA（1，25-（OH）_2D_3）和地塞米松（Dex）处理hPDL细胞和小鼠骨髓细胞（BMCs）共培养时，抗酒石酸酸性磷酸酶（TRAP）阳性多核细胞诱导在富含BMC的培养，但不诱导在低BMC密度培养。而低密度培养的骨髓基质细胞则可诱导破骨细胞的形成 ...更多信息 3）Real-time RT-PCR检测发现牵张后hPDL细胞RANKL mRNA表达明显降低，OPG mRNA表达明显升高。牵张后hPDL细胞培养液中OPG浓度增加，这表明hPDL细胞的拉伸起到抑制BMC中破骨细胞生成的诱导的作用。4）将牵张的hPDL细胞与BMCs在1，25-（OH）_2D_3和Dex存在下共培养，诱导TRAP阳性多核细胞，但数量少于未伸展的hPDL细胞和BMCs共培养中诱导的TRAP阳性多核细胞。RT-PCR结果显示，在含10%FCS的αMEM培养基中培养的hPDL细胞表达血管内皮生长因子（VEGF）mRNA，并且通过使用ELISA在培养基中释放合成的VEGF。6）牵张可增加hPDL细胞VEGF mRNA的表达，牵张培养时细胞培养液中VEGF的浓度高于未牵张培养时。因此，拉伸刺激增强hPDL细胞中VEGF的产生，并可进一步促进PDL组织中的血管生成。少

The present study was carried out to clarify how the human periodontal ligament (hPDL) cells respond to the cyclic stretching. We focused on the role of the stretched hPDL cells for the differentiation of osteoclasts and on the production of vascular endothelial growth factor in the stretched hPDL cells. The results are summarized as follows.1)It was shown by RT-PCR and Northern blotting that cultured hPDL cells express mRNAs of receptor activator of NF-kappa B ligand (RANKL) and its decoy receptor osteoprotegerin (OPG) in αMEM supplemented with 10% FCS in the presence of 1 alpha, 25(OH)__2 vitamin D__3(1, 25-(OH)__2D__3)and dexamethasone(Dex).2)When the coculture of hPDL cells and mouse bone marrow cells (BMCs) were treated with 1, 25-(OH)__2D__3 and Dex, Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells were induced in the BMC-rich culture but not induced in the low BMC-density culture. However, osteoclasts were induced when the low BMC-density culture was treate … More d with an anti-OPG antibody.3)We found that RANKL mRNA was reduced while OPG mRNA was increased markedly by stretching in the hPDL cells by using Real-time RT-PCR The concentration of OPG in the culture media was increased when the hPDL cells were stretched in situ, suggesting that stretching of the hPDL cells acts to suppress induction of osteoclastogenesis in the BMCs.4)When the stretched hPDL cells and BMCs were cocultured in the presence of 1, 25-(OH)__2D__3 and Dex, TRAP-positive multinuclear cells were induced but the number was smaller than that induced in the conculture of unstreched hPDL cells and BMCs.5)It was demonstrated that cultured hPDL cells express vascular endothelial growth factor (VEGF) mRNA in αMEM supplemented with 10% FCS by using RT-PCR, and that the synthesized VEGF was released in the culture media by using ELISA.6)The expression of VEGF mRNA in cultured hPDL cells was increased by stretching and the concentration of VEGF in the media was higher in stretched culture than in unstretched culture. Thus, stretching stimuli enhance production of VEGF in hPDL cells and may further facilitate angiogenesis in the PDL tissue. Less