成牙本质细胞在牙髓免疫反应中作用的研究进展

深龋时龋病相关细菌侵入牙本质深层接近牙髓,成牙本质细胞层位于牙髓牙本质交界处,作为牙髓组织最先接触外源性刺激的第一道防线,可通过模式识别受体(PRRs,如Toll样受体、天然免疫受体NOD)识别多种病原相关分子模式(PAMPs),产生促炎细胞因子、表达细胞粘附分子,启动免疫应答。多种细胞因子、细胞粘附分子参与牙髓炎的发生。本文就成牙本质细胞通过TLRs、NLRs识别细菌性因素、引起牙髓免疫反应导致牙髓炎过程中的作用及其研究进展作一综述。     

Immunoregulatory roles of adhesive interactions between lymphocytes and gingival fibroblasts

Chronic adult periodontitis is usually characterized by inflammatory cell accumulation in the extravascular periodontal connective tissue. In order to reveal how the lymphocyte migration and retention in periodontal lesions is regulated, we have focused on the molecular basis for the adhesive interactions between lymphocytes and human gingival fibroblasts (HGF). In this study, we investigated the involvement of cell adhesion molecules in adhesive interactions between lymphocytes and HGF. We found that activated lymphocytes bound strongly to HGF and VLA integrins, extracellular matrix receptors, play crucial roles in the binding. Interestingly, we first revealed that CD44 molecules (hyaluronate receptor) on lymphocytes also participated in lymphocyte-HGF interactions and that hyaluronate anchored on the surface of HGF functioned as the ligand for CD44. In addition. when HGF were stimulated with inflammatory cytokines such as IL-1. TNFα and IFNγ, the binding avidity between lymphocytes and HGF was significantly increased and the adhesion was mainly mediated by LFA-l/ICAM-1 pathway. We then examined the possibility whether lymphocyte-HGF interaction may cause activation of HGF. When HGF directly interacted with lymphocytes for 3 h, IL-lβ mRNA expression was clearly increased in HGF. These findings suggested that the adhesive interactions between lymphocytes and HGF was mediated at least by VLA integrins, LFA-l/ICAM-1 and CD44/hyaluronate and that the heterotypic cell-cell interactions could mutually cause intracellular signal transduction.     

Autophagic activity and aging in human odontoblasts

Odontoblasts are long-lived post-mitotic cells in the dental pulp, whose function is to form and maintain dentin. The survival mechanisms that preserve the viability of terminally differentiated odontoblasts during the life of a healthy tooth have not been described. In the present study, we characterized the autophagic-lysosomal system of human odontoblasts with transmission electron microscopy and immunocytochemistry, to analyze the mechanisms that maintain the functional viability of these dentinogenic cells. Odontoblasts were found to develop an autophagic-lysosomal system organized mainly by large autophagic vacuoles that are acid-phosphatase-positive to various degrees. These vacuoles expressed the autophagosomal and lysosomal markers LC3 and LAMP2, respectively, in an age-related pattern indicating the organization of a dynamic autophagic machinery. Progressive accumulation of lipofuscin within lysosomes indicates reduced lysosomal activity as a function of odontoblast aging. Our results suggest that autophagic activity in odontoblasts is a fundamental mechanism to ensure turnover and degradation of subcellular components. A reduction in the efficacy of this system might compromise cell viability and dentinogenic secretory capacity. In adult teeth, this condition is described as an 'old odontoblast' stage.     

成牙本质细胞分化的分子机制

成牙本质细胞的分化是一个由多种分子参与调控的复杂过程，涉及到生长因子、受体分子、信号分子、转录因子、牙本质基质蛋白等分子的信号转导网络。本文对其信号转导各个环节的研究进行了概述。     

Bradykinin mediates phosphorylation of eNOS in odontoblasts

While the activation of eNOS by Akt/PKB-dependent phosphorylation, leading to NO release, and the inhibition of enzyme activity by bradykinin (BK)-mediated phosphorylation of eNOS in endothelial cells are established, the phosphorylation of eNOS in odontoblasts is unknown. To clarify the regulation of eNOS in odontoblasts by BK, we examined the phosphorylation of eNOS, Akt/PKB, and ERK1/2 in odontoblasts of rat molars. BK (10(-7) M) transiently induced the phosphorylation of eNOS at Ser1177, Akt/PKB in odontoblasts, while it induced the phosphorylation of eNOS at Thr495 throughout the entire period of BK treatment. BK receptor 2 antagonist HOE 140 (10(-6) M) significantly reduced signal intensities of phosphorylated-eNOS at Ser1177, Thr495, and phosphorylated-Akt/PKB. These results suggest that BK has dual effects on the activation of eNOS in odontoblasts, the Akt/PKB-dependent up-regulation of eNOS by the transient phosphorylation at Ser1177, and the ERK1/2-independent down-regulation of eNOS by the phosphorylation at Thr495.     

Immunocytochemical detection of apoptosis in human odontoblasts

Pulpal chamber size decreases on ageing due to primary and secondary dentin deposition. This work was designed to find out the consequences of this pulp chamber reduction on odontoblast number and distribution. Twenty-one healthy human premolars were equally divided into three groups from 11-, 12.5- and 14-yr-old adolescents, respectively). The external and the internal perimeters of dentin were recorded on vestibulo-lingual sections, from buccal to lingual cemento-enamel junction using an image analysis system. Nuclei of the odontoblasts were recorded on 12 automatically selected fields. On nine erupted premolars (3 teeth from each 11-, 12.5- and 14-yr-old patients), apoptosis was detected by confocal microscopy using a modification of the original TUNEL method. Apoptotic cells were labeled in central pulp fibroblasts, perivascular endothelial cells, and in odontoblasts. When the pulp volume decreases due to primary dentin production, the decrease of the surface available for odontoblasts is compensated for by a multilayer distribution of cells. Secondary dentin deposition, associated with odontoblasts reorganization in a single layer, results in a hyperbolic decrease of the odontoblasts number. This decrease seems to result from a programmed cell death, which eliminates half of the odontoblasts over a 4-yr period.     

Mitochondrial autophagy and lipofuscin accumulation in aging odontoblasts

Aging of long-lived post-mitotic cells is characterized as a progressive and irreversible reduction of functional activity. In such cells, mitochondria are organelles critical for bioenergetic supply, whose turnover is mediated by an autophagic-lysosomal pathway. In human teeth, odontoblasts are post-mitotic cells responsible for sensory function and dentin preservation. Here, human odontoblasts were processed for immunohistochemistry with antibodies against mitochondrial (MTCO2) and lysosomal (LAMP2) markers, and comparatively analyzed in two age groups (young-adult and adult) with light and electron microscopy. Selective engulfment of mitochondrial profiles into autophagic vacuoles is common in young-adult odontoblasts, suggesting a microautophagic pathway. With age, the odontoblast layer is reduced in width, and mitochondrial elements converge around large clusters of autofluorescent lipofuscin deposits. Age-related changes in odontoblasts are observed as a long-term process in which the progressive accumulation of intralysosomal debris limits the autophagic turnover of mitochondrial components, causing an eventual decline in physiological cell functions, which leads to increased vulnerability under stress conditions.     

Effect of vincristine on odontoblasts in rat incisor

abstract — Vincristine in doses of 0.1, 0.3, 0.5 and 0.7 mg/kg was administered to 60 rats in four groups. Histomorphologic investigation of the odontoblast population in the maxillary incisors revealed dose-dependent reactions consisting of (1) swelling of the odontoblasts and an accumulation of abnormal mitotic cells in the germinative parts of the pulp after 5 h, (2) a supervening necrosis and destruction of some odontoblasts and of the mitotic cells after 24 h, and (3) after 3 d, a reversal to normal structure in some parts of the odontoblast population, but a further development of the vincristine-induced changes, with severe cellular derangement and irregular predentin production, in others.     

成牙本质细胞的基本特征及其研究进展

成牙本质细胞作为牙髓细胞的主要成分，具有在牙发育期间和成熟牙内生成牙本质的功能。目前的研究多基于牙髓细胞的二维体外培养，随着对牙髓组织研究的不断深入，牙髓细胞体外三维立体模型的建立将是未来研究的热点。作者回顾了近年来对成牙本质细胞的研究情况，主要阐述了成牙本质细胞的形态、超微结构、功能、蛋白表达的特征及其研究进展和今后的研究方向。     

Isolation of primary odontoblasts: Expectations and limitations

The purpose of this study was to evaluate different protocols of enzymatic treatment (collagenase with either protease, trypsin or hyaluronidase) to isolate mature odontoblasts. Primary odontoblasts were obtained from human molars, which was confirmed by histology and scanning electron microscopy. The combination of collagenase with protease appeared most suitable and resulted in higher cell numbers and better integrity of the odontoblast processes, whereas combination with hyaluronidase or trypsin led to truncated processes and detachment of cell patches instead of single cells. However, trypan blue staining after 24 h showed that odontoblasts in culture did not remain viable. Gene expression analysis was possible after mRNA extraction from tissues ex vivo and real‐time semi‐quantitative PCR revealed increased expression of collagen, nestin, bone sialoprotein and dentin matrix acidic phosphoprotein 1 in the odontoblast layer. Though primary odontoblasts could not be cultivated after isolation, characteristic genes were identified to differentiate odontoblasts from pulp fibroblasts.     

Autoradiography of [3H]-proline uptake by fibroblasts of the periodontal ligament and odontoblasts of the rat incisor after guanethidine-induced sympathectomy

The uptake of [3H]-proline was monitored by quantitative autoradiography at 30, 45 and 90 days of age. Grain counts in sympathectomized rats did not vary significantly from control values at any time. Thus the sympathetic nervous system does not influence the metabolic activity of these cells, which agrees with morphologic evidence that the innervation of the rat incisor may not be autonomic.     

Exostosin 1 is expressed in human odontoblasts

ObjectiveDental pulp is soft connective tissue maintaining the vitality of the tooth, while odontoblasts form the dentin. Our earlier DNA microarray analysis revealed expression of putative tumour suppressor exostosin 1 (EXT-1) in odontoblasts. EXT-1 is essential for heparan sulphate synthesis, which may play a role in the dentin mineralization. Since the absence of the functional EXT-1 causes bone tumours, expression in odontoblasts is interesting. Our aim was to analyse further the EXT-1 expression in human tooth.DesignsDNA microarray and PCR techniques were used to study the EXT-1 expression in mature native human odontoblasts and pulp tissue as well as in newly-differentiated cultured odontoblast-like cells. Immunohistochemistry was performed to study EXT-1 protein in mature human teeth, teeth with incomplete root and developing teeth.ResultsMarkedly higher EXT-1 was observed in mature odontoblasts than in pulp at mRNA level with DNA microarray and PCR techniques. Immunohistochemistry of mature tooth revealed EXT-1 both in odontoblasts and the predentin but not in the dentin. EXT-1 was also observed in the odontoblasts of incomplete root, but the localization of the staining was different. In developing foetal tooth, staining was detected in ameloblasts and the basal lamina.ConclusionsThe detection of EXT-1 in both mature and newly-differentiated cells indicates a role in the odontoblast function, and EXT-1 staining in the predentin indicates a function in the dentin formation. Detection of EXT-1 in developing teeth indicates a role in tooth development.