Tumor Necrosis Factor-α Induces Matrix Metalloproteinases-3,-10, and-13 in Human Periodontal Ligament Cells
Tumor Necrosis Factor-α Induces Matrix Metalloproteinases-3,-10, and-13 in Human Periodontal Ligament Cells
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DOI：
10.1902/jop.2013.130063
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发表时间：
2014-03-01
影响因子：
4.3
通讯作者：
Park, Sang Hyuk
中科院分区：
文献类型：
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作者：
Ahn, Su-Jin;Rhim, Eun-Mi;Park, Sang Hyuk
背景：细胞因子可以诱导多种与牙周组织破坏有关的生物介质，包括基质金属蛋白酶（MMP）。已知 MMP 会降解牙周膜附着，骨基质蛋白和金属蛋白酶组织抑制剂 (TIMP) 会抑制 MMP 的活性。本研究的目的是在体外研究肿瘤坏死因子 (TNF)-a 对人牙周膜 (PDL) 细胞中 MMP 表达的影响，并确定哪些 MMP 在该刺激下特异性表达。方法：用 TNF-a 刺激培养的 PDL 细胞，并用 MMP 抗体阵列进行分析。使用实时聚合酶链反应 (PCR)、酶联免疫吸附测定 (ELISA) 以及使用细胞裂解物和酶谱进行的蛋白质印迹来测量 MMP-3、-10 和 -13 的信使 RNA (mRNA) 和蛋白质水平。为了检查TNF受体(TNFR)的表达，通过流式细胞术检查PDL细胞，并在用拮抗剂阻断TNFR后观察MMP-3、-10和-13的表达。通过配对t检验分析实时PCR、ELISA和蛋白质印迹的结果。结果：抗体阵列显示TNF-α刺激上调最强烈的蛋白是MMP-3，其次是MMP-13和MMP-10。 TNF-α受体阻断剂特异性抑制MMP-3和-13的表达。此外，TNF-a 增加了 MMP-3、-13 和 -10 中 MMP mRNA 的水平（按降序排列）。然而，ELISA 显示 MMP-13 是上调最多的蛋白质，其次是 MMP-10 和 MMP-3。 Western blotting表明TNF-a增加了MMP-3和-13的水平，但对MMP-10的水平没有显着影响，酶谱显示TNF-a增加了所有形式的MMP-3和-13的活性，但没有检测到MMP-10。流式细胞术表明大多数 PDL 细胞表达 TNFR1。结论：TNF-a (10 ng/mL) 上调人 PDL 细胞中 MMP-3、-10 和 -13 的水平。这些结果表明这些蛋白质在 PDL 炎症中发挥重要作用。
Background: Various biologic mediators, including matrix metalloproteinases (MMPs), that are implicated in periodontal tissue breakdown can be induced by cytokines. MMPs are known to degrade periodontal ligament attachment, and bone matrix proteins and tissue inhibitors of metalloproteinase (TIMPs) inhibit the activity of MMPs. The aim of this study is to investigate the effect of tumor necrosis factor (TNF)-a on the expression of MMPs in human periodontal ligament (PDL) cells in vitro and establish which MMPs are expressed specifically in response to that stimulus.Methods: Cultured PDL cells were stimulated with TNF-a and analyzed with an MMP antibody array. Real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and western blot with cell lysate and zymography were used to measure messenger RNA (mRNA) and protein levels of MMP-3, -10, and -13. To examine TNF receptor (TNFR) expression, PDL cells were examined by flow cytometry, and expression of MMP-3, -10, and -13 was observed after blocking the TNFR with an antagonist. Results from real-time PCR, ELISA, and western blot were analyzed by paired t test.Results: The antibody array showed that the protein most strongly upregulated by TNF-a stimulation was MMP-3, followed by MMP-13 and MMP-10. The TNF-a receptor blocker specifically inhibited expression of MMP-3 and -13. In addition, TNF-a increased levels of MMP mRNAs in MMP-3, -13, and -10 (in decreasing order). However, ELISAs showed that MMP-13 was the most upregulated protein, followed by MMP-10 and MMP-3. Western blotting indicated that TNF-a increased MMP-3 and -13 levels but had no significant effect on the level of MMP-10, and zymography showed that TNF-a increased the activities of all forms of MMP-3 and -13, but MMP-10 was not detected. Flow cytometry demonstrated that the majority of PDL cells expressed TNFR1.Conclusions: TNF-a (10 ng/mL) upregulates levels of MMP-3, -10, and -13 in human PDL cells. These results suggest that these proteins play an important role in the inflammation of PDLs.