Dentine matrix proteins: isolation and effects on human pulp cells
Dentine matrix proteins: isolation and effects on human pulp cells
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DOI：
10.1111/iej.12754
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发表时间：
2018-05
影响因子：
5
通讯作者：
M. Widbiller;A. Eidt;S. R. Lindner;K. Hiller;H. Schweikl;Wolfgang Buchalla;K. Galler
中科院分区：
文献类型：
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作者：
M. Widbiller;A. Eidt;S. R. Lindner;K. Hiller;H. Schweikl;Wolfgang Buchalla;K. Galler
目的建立一种简便、高效的人牙本质基质蛋白的分离和浓缩方法，并研究牙本质基质蛋白对人牙髓细胞行为的影响。方法从人牙本质中分离基质蛋白，纯化、浓缩并用蛋白质和酶联免疫吸附测定（ELISA）进行表征。培养基中补充了不同浓度的Eclase，称为Eclase 1-10 000，通过DNA和MTT测定评估人牙髓细胞的活力和增殖;通过流式细胞术定量细胞凋亡事件。在改良的Boyden室测定中评估了依地平的趋化作用。实时荧光定量PCR检测牙髓细胞成牙本质细胞标志基因的表达水平，茜素红染色观察成牙本质细胞的矿化能力。进行非参数统计分析，以在所有时间点成对比较不同组（Mann-Whitney U检验，α = 0.05）。结果高浓度的依替米松作用5 d（依替米松1000）和7 d（依替米松500）后显示出显著的抗增殖作用（P ≤ 0.023），而不影响细胞活力。细胞凋亡几乎不受影响（P ≥ 0.089）。在低剂量时，依地平对牙髓细胞产生浓度依赖性的趋化刺激，具有统计学意义（在依地平10时，P = 0.006）。基因表达的变化表明成牙本质细胞样细胞的分化，这是由矿物结节形成的结果所证实。结论：本研究为牙本质基质蛋白的分离和浓缩提供了一种新的、有效的、省时的方法。由于内皮素刺激趋化性、分化和矿化而不影响活力，内源性牙本质基质蛋白可能对牙髓再生或工程化的方法有价值。
AIM To establish a simplified and efficient protocol for the isolation and concentration of matrix proteins from human dentine, and to assess the effects of extracted dentine matrix proteins (eDMP) on the behaviour of human pulp cells. METHODOLOGY Matrix proteins were isolated from human dentine, purified, concentrated and characterized with protein and enzyme-linked immunosorbent assays (ELISA). Culture media were supplemented with eDMP in different concentrations, referred to as eDMP 1-10 000, to assess viability and proliferation of human pulp cells by DNA and MTT assays; apoptotic events were quantified by flow cytometry. Chemotactic effects of eDMP were assessed in a modified Boyden chamber assay. Expression levels of odontoblastic marker genes in pulp cells cultured with eDMPs were determined by real-time quantitative PCR, and the ability to induce mineralization was demonstrated by alizarin red staining. Nonparametric statistical analysis was performed to pairwise compare different groups at all time-points (Mann-Whitney U-test, α = 0.05). RESULTS High concentrations of eDMP exhibited significant antiproliferative effects (P ≤ 0.023) after 5 (eDMP 1000) and 7 days (eDMP 500) without affecting cell viability. Apoptosis was barely influenced (P ≥ 0.089). eDMP exerted a concentration-dependent chemotactic stimulus on dental pulp cells with statistical significance already at low dosage (P = 0.006 at eDMP 10). Changes in gene expression indicated a differentiation into odontoblast-like cells, which was corroborated by findings of mineral nodule formation. CONCLUSIONS A novel, effective and time-saving protocol for isolation and concentration of dentine matrix proteins is presented. As eDMP stimulates chemotaxis, differentiation and mineralization without affecting viability, endogenous dentine matrix proteins might be valuable for approaches to regenerate or engineer dental pulp.