大鼠牙胚组织发育过程中釉原蛋白mRNA的表达

目的：观察釉原蛋白（Ａｍ）基因在大鼠牙胚组织发育过程中的表达。方法：采用原位杂交的方法,检测大鼠孕期１７、１８、１９ｄ和出生后１、３、５、７、９ｄ牙胚中成釉细胞ＡｍｍＲＮＡ的表达。结果：从出生后１ｄ至出生后９ｄ大鼠第一磨牙牙胚中均检测到ＡｍｍＲＮＡ的表达,其中出生后第５ｄ表达最高,成釉细胞分泌完成后Ａｍ的表达下降。成牙本质细胞在发育的任何时期均未见到Ａｍ的表达。结论：釉原蛋白基因的转录只发生在牙胚发育的早期,前成釉细胞极化阶段和成釉细胞分泌阶段;发育成熟的釉基质中没有釉原蛋白。成牙本质细胞不表达釉原蛋白基因。     

Comparative gene expression profile analysis between native human odontoblasts and pulp tissue

Aim  To undertake a large-scale analysis of the expression profiles of native human pulp tissue and odontoblasts, and search for genes expressed only in odontoblasts.
Methodology  Microarray was performed to pooled pulp and odontoblasts of native human third molars and to pooled +/− TGF-β1 cultured pulps and odontoblasts (137 teeth). The repeatability of microarray analysis was estimated by comparing the experimental pulp samples with expression profiles of two pulp samples downloaded from the GEO database. The genes expressed only in the experimental pulp samples or in odontoblasts were divided into categories, and the expression of selected odontoblast-specific genes of extracellular matrix (ECM) organization and biogenesis category was confirmed with RT-PCR and Western blot.
Results  A 85.3% repeatability was observed between pulp microarrays, demonstrating the high reliability of the technique. Overall 1595 probe sets were positive only in pulp and 904 only in odontoblasts. Sixteen expressed sequence tags (ESTs), which represent transcribed sequences encoding possibly unknown genes, were detected only in odontoblasts; two consistently expressed in all odontoblast samples. Matrilin 4 (MATN4) was the only ECM biogenesis and organization related gene detected in odontoblasts but not in pulp by microarray and RT-PCR. MATN4 protein expression only in odontoblasts was confirmed by Western blot.
Conclusions  Pulp tissue and odontoblast gene expression profiling provides basic data for further, more detailed protein analysis. In addition, MATN4 and the two ESTs could serve as an odontoblast differentiation marker, e.g. in odontoblast stem cell research.     

TLR4 mediates LPS-induced VEGF expression in odontoblasts

Lipopolysaccharide (LPS) from gram-negative bacteria cell walls such as Prevotella intermedia and Escherichia coli induce vascular endothelial growth factor (VEGF) expression in odontoblasts, but not in undifferentiated dental pulp cells. CD14 and TLR4 are responsible for LPS signaling in macrophages, but their expression levels and function in dental pulp cells are unknown. We showed here that murine odontoblast-like cells (MDPC-23) express CD14 and TLR4 by immunohistochemistry and flow cytometry. In contrast, undifferentiated dental pulp cells (OD-21) presented low or no expression of these two receptors. MDPC-23 cells showed CD14 and TLR4 up-regulation upon exposure to LPS, as determined by real time PCR. Dominant negative murine TLR4 (DN-mTLR4) transfected MDPC-23 cells did not show upregulated VEGF expression in response to LPS stimulation. These results demonstrate that odontoblast-like cells express CD14 and TLR4, and that LPS-induced VEGF expression is mediated, at least in part, by TLR4 signaling.     

热休克蛋白70在两个年龄组前磨牙成牙本质细胞表达的比较研究

目的观察热休克蛋白(HSP)70在前磨牙成牙本质细胞表达的增龄变化特点。方法采用免疫组化法结合图像分析系统,对HSP70在年老组(71~80岁)和年轻组(13~20岁)前磨牙成牙本质细胞和成牙本质细胞胞质中的表达强度进行比较。结果年老组成牙本质细胞内总的HSP70免疫组化染色的平均吸光度值比年轻组降低,而胞质内HSP70免疫组化染色的平均吸光度值平均值比年轻组略高,但无显著性差异。结论年老患者成牙本质细胞抵御急性刺激的能力下降可能与胞核内HSP70表达强度下降有关。     

DMP1-targeted Cre expression in odontoblasts and osteocytes

Odontoblasts in dentin and osteocytes in bone contain dendritic processes. To test if their dendrites share a common feature, we compared their cellular morphology as visualized using scanning electron microscopy. Analysis of our data showed that both cells share an identical dendritic canalicular system and express extensive processes forming a complex network within the mineralized matrix. Because dentin matrix protein 1 (DMP1), an extracellular matrix protein, is highly expressed in both types of cells, we next tested, using a transgenic approach, whether a 9.6-kb Dmp1 promoter-4-kb 1st intron would be able to target Cre cDNA in these cells for expression/deletion of other genes in odontoblasts and osteocytes. We determined the specificity and efficiency of Cre activity by crossing Dmp1-Cre mice with ROSA26 reporter mice. Results showed that odontoblasts and osteocytes were specifically targeted, suggesting that this animal model will be useful for the preferential study of gene functions in both types of cells.     

体外培养人牙周牙髓相关细胞中釉原蛋白的表达

目的:观察釉原蛋白(amelogenin,Am)基因在体外培养的人牙龈上皮细胞及口腔外胚间充质来源细胞(人牙龈成纤维细胞、人牙周膜成纤维细胞和人牙髓细胞)中的表达.方法:采用反转录多聚酶链式反应(RT-PCR)技术,检测培养细胞中釉原蛋白mRNA的表达.采用蛋白质免疫印迹技术检测培养细胞中釉原蛋白的表达.结果:培养的人牙周膜成纤维细胞、牙髓细胞、牙龈成纤维细胞和牙龈上皮细胞中均未检测到釉原蛋白及其mRNA的表达.结论:体外培养的人牙周膜成纤维细胞、牙髓细胞、牙龈成纤维细胞和牙龈上皮细胞不表达釉原蛋白基因.     

热休克蛋白70在第三磨牙成牙本质细胞中表达的增龄变化

目的观察热休克蛋白70（heat shock protein70 HSPT0）在第三磨牙成牙本质细胞中表达的增龄变化。方法共选取33例21—30岁、31～40岁、4I～50岁和51～60岁四个年龄组健康第三磨牙的组织标本进行免疫组化染色,并通过计算机图像分析对比四组牙髓组织成牙本质细胞中HSP70表达量的不同。结果41～50岁和51～60岁年龄组平均光密度值明显低于21～30岁年龄组和31～40岁年龄组（P〈0．05）,其余各组间无显著性差异。结论以41～50岁牟龄组为转折点,HSP70在第三磨牙成牙本质细胞中的表达强度显著下降。     

成牙本质细胞中上游刺激因子1对骨桥素表达调控的研究

目的检测成牙本质细胞中上游刺激因子1（upstream stimulatory factor 1，USF1）对骨桥素表达的影响。方法培养成牙本质细胞MDPC-23，稳定转染PCMV-USF1和酸性-USF（A-USF）质粒，提取总RNA，半定量反转录聚合酶链反应检测骨桥素的表达水平，并对结果进行统计学分析。结果筛选出稳定转染USF1和A-USF的抗性克隆，USF1、A-USF转染组和对照组骨桥素相对灰度比值分别为：60．33％±4．51％、229．33％±7．09％、110．00％±15．62％，转染组与对照组差异有统计学意义（P〈0．01）。结论USF1可调控成牙本质细胞骨桥素mRNA转录，该作用被A-USF部分阻断。     

Spatiotemporal expression of sclerostin in odontoblasts during embryonic mouse tooth morphogenesis

Introduction

Sclerostin is the product of the SOST gene. Loss-of-function mutations in the SOST gene result in a high bone mass phenotype, thus confirming that sclerostin is a negative regulator of bone mass. SOST knockdown in humans also causes oral and dental malformations. However, the relationship between sclerostin and tooth development is unclear.

Methods

Using immunohistochemical techniques, we investigated sclerostin expression during fetal mouse tooth development and adult mouse tooth morphogenesis.

Results

Sclerostin was expressed in the secretory odontoblasts located along the ameloblasts of fetal mouse tooth germ and adult incisor. Sclerostin expression was also observed in the fetal and adult osteocytes in the jaw bone.

Conclusion

These results suggest that sclerostin, one of the important regulatory factors of differentiated odontoblast function, may usable in vital pulp therapy.     

Gene expression in a pure population of odontoblasts isolated by laser-capture microdissection.

Studies of odontoblast differentiation and function have been limited due to difficulties in obtaining sufficient numbers of intact cells. We describe a novel approach of laser-capture microdissection to obtain homogenous populations of pre-odontoblasts and odontoblasts from tissue sections of mouse molar cusp tips. Fixation, processing, and staining conditions were assessed for the optimal retrieval of total RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of alpha1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA from three-day-old captured odontoblasts was sufficient in quantity and quality. Odontoblast-specific gene expression was studied by RT-PCR analysis performed in a single streptavidin-coated tube. At E15.5, Days 0 and 3, gene expression in laser-captured odontoblasts resembled that seen in vivo by in situ hybridization. The use of LCM is thus a valuable means of retrieving quality RNA from discrete populations of odontoblasts at different stages of dentinogenesis.     

Alpha v beta 3 integrin expression in human odontoblasts and co-localization with osteoadherin

Integrins are heterodimeric transmembrane receptors which promote cell adhesion, thus contributing to the maintenance of tissue organization in both normal and pathological conditions. To characterize the way odontoblasts may interact with other cells and the extracellular matrix in human teeth, we studied expression of alpha v beta 3 integrin, a putative receptor for osteoadherin. We showed that alpha v beta 3 integrin expression was restricted to odontoblasts, blood vessels, and small rounded cells in sound and carious pulp. Odontoblast staining intensity increased from the apical to the cusp region. Osteoadherin staining was strong in the whole odontoblast layer (with a slight decrease in the cusp region) and in predentin. Odontoblasts differentiating in vitro were stained with the anti-alpha v beta 3 integrin antibody, first at the level of intercellular contacts, then throughout the cell membrane. These results suggest that the alpha v beta 3 integrin could play a role in interodontoblast adhesion and odontoblast binding to the surrounding predentin/dentin/pulp matrix, possibly through osteoadherin.     

猪发育期牙胚釉原蛋白的分离纯化及其多克隆抗体的制备

目的　制备猪釉质蛋白多克隆抗体,为釉原蛋白的检测提供条件。方法　选择1月龄乳猪,分离埋于上下颌骨内的牙胚,刮取牙胚表面干酪样尚未完全矿化的釉质基质,通过盐酸胍抽提及SephadexG-200柱层析分离纯化猪发育期牙胚釉原蛋白,联合免疫家兔,抗血清经DE-52纤维素纯化,并经ELISA测定抗体效价。结果　 SDS-PAGE电泳结果发现采用SephadexG-200葡聚糖凝胶过滤能达到较为理想的釉原蛋白分离纯化效果,用所提纯的猪釉原蛋白免疫家兔,成功地制得兔抗猪釉原蛋白抗血清,抗血清稀释达1∶32 000时,采用ELISA法仍有明显的抗原抗体反应。结论　本实验成功制备得到抗釉原蛋白多克隆抗体,为釉原蛋白的检测提供了条件。     

Toll样受体4在人成牙本质细胞和牙髓组织中的表达

目的 探讨人牙髓组织和成牙本质细胞中Toll样受体4（TLR4）的表达与分布特点，及其在牙髓组织天然免疫防御中的可能作用。方法从新鲜拔除的人第三磨牙获取牙髓组织和成牙本质细胞，扫描电镜观察牙髓摘除后髓腔壁成牙本质细胞形态和附着情况；RT—PCR检测人牙髓组织以及成牙本质细胞中TLR4mRNA的表达情况：采用免疫印迹技术和免疫组织化学方法检测TLR4蛋白的表达与定位。结果扫描电镜观察牙髓摘除后成牙本质细胞大部存留在髓室壁上，细胞形态良好：RT-PCR结果发现健康牙髓、成牙本质细胞均可表达TLR4mRNA，产物大小为278bp；免疫印迹法检测正常牙髓、成牙本质细胞在相对分子质量89000附近出现蛋白条带；免疫组化显示牙髓组织中的成牙本质细胞、血管内皮细胞TLR4免疫反应阳性。正常牙髓中牙髓成纤维细胞不表达TLR4。结论成牙本质细胞通过表达TLR4参与牙髓牙本质复合体的先天免疫反应，TLR4在牙髓抵御外来病原微生物入侵的宿主免疫防御中起重要作用。     

HUGO (FNDC3A): a new gene overexpressed in human odontoblasts

Previously, we established a subtractive cDNA library enriched in odontoblast-specific genes and hypothesized that new, previously unidentified, markers would be present, associated with the odontoblast phenotype. In this paper, we report the first characterization of a new gene we have named HUGO, and its associated deduced protein sequence. This gene expression is under the control of two alternative promoters, resulting in the synthesis of two proteins, one of which, HUGO2, is included in the other, HUGO1. HUGO proteins are mainly composed of a proline-rich region at the N-terminus, 8 type III-fibronectin modules, and a transmembranous helix at the C-terminus. In odontoblasts, the proteins are located in Golgi vesicles. However, they display a broader expression pattern, since they are also expressed by nerve fibers in the dental pulp and other tissues (e.g., trachea, brain, kidney), as demonstrated by immunohistochemistry and qPCR, respectively. Their location in odontoblasts suggests a role in collagen and glycosaminoglycan synthesis.     

Amelogenin: lessons from evolution

Amelogenin plays a crucial role in enamel structure and mineralization, but the function of its various domains is far to be understood. Evolutionary analysis seems to be a promising way to approach structure/function relationships. In this paper, we review the knowledge of amelogenin with a particular focus on what we have learnt from evolution, and we bring new data on the origin and evolution of this molecule. The comparison of amniote (reptiles and mammals) amelogenin sequences reveals that, in contrast to the well-conserved C- and N-terminal domains, the central region (most of exon 6) is highly variable. The evolutionary analysis indicates that it was created by repeated insertion of three amino acids (triplets ProXGlu or ProXX). In several mammalian lineages a new run of triplet insertions and deletions has occurred independently in a locus considered a hot spot of mutation for mammalian amelogenin. In lizard and snake amelogenin evolves rapidly. Sequence alignment reveals that several residues in the N- and C-terminal regions were kept unchanged during 250 million years (MY), proving their importance for amelogenin structure and function. This alignment permits a rapid validation of the amelogenin mutations in human. Genome sequencing and gene mapping permitted to refine the amelogenin story, in relation to the common location (chromosome 4 in human) of several genes coding for dental proteins and SPARCL1, a SPARC (osteonectin) relative. Amelogenin shares a similar organisation with these genes and a blast search in databanks indicates a strong relationship between amelogenin, ameloblastin and enamelin. Taken together these data suggest that amelogenin could have originated from either ameloblastin or enamelin, themselves being created from SPARCL1, which itself originated from a SPARC duplication, 600 millions years ago.     

Molecular expression of Mg2+ regulator TRPM7 and CNNM4 in rat odontoblasts

ObjectiveMagnesium, the second most abundant cation in cellular fluid, is critical for mineralization of hard tissues. Among the molecules involved in cellular Mg²⁺ homeostasis, functional impairment of Mg²⁺ permeable ion channel TRPM7 or Mg²⁺ transporter CNNM4 have been found to result in severe hypomineralization of the enamel and dentin. However, molecular expressions of TRPM7, CNNM4 and their respective homologues have not been fully investigated in adult odontoblasts.DesignExpressions of TRPM6, TRPM7, CNNM1, CNNM2, CNNM3, CNNM4 were screened in acutely dissociated rat odontoblasts by single cell RT-PCR. Among these candidates, expression levels of TRPM7 and CNNM4 were compared along the odontoblast layer by immunohistochemical analysis. Finally, the coexpression pattern of TRPM7 and CNNM4 in subcellular regions was examined by immunocytochemical analysis.ResultsScRT-PCR revealed high expression rate of TRPM7 and CNNM4 in odontoblasts, with CNNM4 detected almost exclusively in TRPM7-positive odontoblasts. However, CNNM2 and CNNM3 were detected in only a small population of odontoblasts, and TRPM6 and CNNM1 were not detected even in the pulp tissue. Immunohistochemical analysis revealed higher CNNM4 expression in the apical odontoblast layer than the coronal area, in contrast to the ubiquitous expression of TRPM7. Lastly, immunocytochemical analysis revealed colocalization of CNNM4 with TRPM7 in the odontoblastic process.ConclusionsCNNM4 and TRPM7 may serve as main Mg²⁺ regulators in odontoblasts, possibly with selective involvement of CNNM4 in apical dentin formation or mineralization. Colocalization of TRPM7 and CNNM4 in the odontoblastic process suggest functional coupling of these two molecules to maintain Mg²⁺ homeostasis.