Mechanisms of mineralization on Dental Pulp Using Control of Differentiation Programs and Re-Programming

牙髓组织中存在大量间充质细胞，这些细胞在各种刺激下可分化为具有硬组织形成能力的细胞。本研究的目的是阐明牙髓硬组织形成的机制。该细胞通过从正常牙髓中长出而制备，所述正常牙髓是从正畸治疗期间在无菌条件下从20岁患者的第三磨牙中提取的。患者在提供样本前提供知情同意书。牙髓细胞经50 μM抗坏血酸、10 mMβ-甘油磷酸盐、100 nM地塞米松处理后，再经1 mM 5-氮杂胞苷处理后，ALP活性明显高于未处理的牙髓细胞。5-氮胞苷对牙髓细胞有去分化作用。牙髓细胞中可能存在大量具有矿化活性的诱导细胞。5-氮杂胞苷对细胞进行去甲基化，使细胞分化。结果发现，在牙髓细胞刺激形成硬组织的过程中，有多种基因参与，HDP细胞在含或不含50 mg/ml EMD的培养液中，提取mRNA。将荧光标记的样品在人cDNA微阵列（Clontech，1，100个基因）上杂交，并且使用阵列扫描仪测量每个基因的荧光强度。从EMD牙髓细胞基因表达变化的分析结果来看，血清诱导激酶、细胞周期蛋白依赖性激酶2和IGF-1等基因的mRNA表达水平升高。本研究中发现的基因可能参与细胞增殖和钙化过程。基因表达谱芯片技术有助于了解HDP细胞的生物学效应。

Many mesenchyme cells exist in the dental pulp tissue, these cells may be differentiated to capable of hard tissue formation by various stimuli. The purpose of this study is the mechanisms that the mechanisms of hard tissue formation on dental pulp is clarified. The cells were prepared by outgrowth from normal dental pulp obtained from the third molar extracted under aseptic conditions from a 20-year-old patient during orthodontic treatment. The patient gave informed consent before providing the sample. After the dental pulp was extracted, the tissue was minced, placed on a 35-mm^2 tissue-culture dish and then covered with a sterile glass coverslip.ALP activity of dental pulp cells which were treated with 50 μM ascorbic acid, 10 mMβ-glycerophosphate, 100 nM dexamethasone after treated with 1 mM 5-Azacytidine was more strongly, than none treated 5-Azacytidine. It seemed that the dental pulp cells were de-differentiated by 5-Azacytidine. It may be possible that a number of dental pulp cells were induced cells with activity of mineralization. It seems that by 5-Azacytidine made de-metylation to the cells and many cells were differentiated. It is found that many gene when dental pulp cells were stimulated to hard tissue formation.HDP cells were incubated with/without 50 mg/ml EMD and mRNA were extracted. The fluorescent labeled samples were hybridized on a human cDNA microarray (Clontech, 1,100 genes ), and the fluorescence intensity of each gene was measured using an array scanner. From the analytical results of gene expression fluctuation of pulp cell with EMD, many genes increased their mRNA levels including serum iducible kinase, cycline dependent kinase 2 and IGF-1. Identified genes found in this study may involved in cell proliferation and calcification process. Gene expression profiling using cDNA microarray shoud be useful understanding the biological effect to HDP cells.