RESUMO
The odontoblast phenotype has been mainly approached by the biochemical characterization of dentin matrix proteins and by extrapolation of the knowledge of bone cell biology, since dentin and bone share many similarities. In fact, direct investigations of the odontoblast phenotype have been hindered by the limited number of cells within the dental pulp and the difficulty in microdissection and isolation of a pure population of these cells. To overcome these obstacles, we previously developed a cell-culture system that promotes differentiation of human dental pulp cells into odontoblasts. This material now permits the study of odontoblasts through molecular biology techniques. Therefore, we constructed a cDNA library enriched for odontoblast-specific genes using the suppression subtractive hybridization technique (SSH). This library led us to identify new genes expressed by odontoblasts. In this paper, we will focus on some genes implied in various functions associated with odontoblast differentiation, such as cell polarization (MAP1B), dentin mineralization (PHEX, osteoadherin), and relationships between odontoblasts and nerve cells (reelin). Another important fact is that about 40% of the cDNA were unknown genes. Therefore, one can speculate that some of them will be odontoblast-specific, since, until now, only one gene (DSPP) presents this characteristic.
Assuntos
Dentinogênese/genética , Odontoblastos/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Técnicas de Cultura de Células , Diferenciação Celular/genética , Polaridade Celular/genética , Polpa Dentária/citologia , Proteínas da Matriz Extracelular/genética , Biblioteca Gênica , Humanos , Hibridização Genética/genética , Microcorpos/genética , Proteínas Associadas aos Microtúbulos/genética , Biologia Molecular , Proteínas do Tecido Nervoso/genética , Endopeptidase Neutra Reguladora de Fosfato PHEX , Fenótipo , Fosfoproteínas/genética , Precursores de Proteínas/genética , Proteínas/genética , Proteoglicanas/genética , Proteína Reelina , Serina Endopeptidases , Sialoglicoproteínas , Supressão Genética/genética , Calcificação de Dente/genéticaRESUMO
Odontoblasts are highly specialized cells aligned at the edge of the dental pulp. As a step towards understanding the complex mechanisms underlying their terminal differentiation, the gene expression pattern was examined in human cultured odontoblast cells. Suppression substractive hybridization (SSH) was used to establish a substracted cDNA library specific for human odontoblasts. For this purpose, cDNAs from human cultured fibroblastic pulp cells were substracted to cDNA from human cultured odontoblasts. The nucleotide sequence of 154 substracted cDNA clones was determined. We identified 130 preferentially expressed gene fragments in odontoblasts as compared with the fibroblastic pulp cells. Ten of them were already identified in odontoblasts such as DSPP, BSP, enamelysin and Col1A1. We confirmed their overexpression by RT-PCR on the cultured cells and in vivo by in situ hybridization on human molars. Another 64 clones corresponded to known genes. Among them, two clones were of particular interest: reelin, which was first detected in the brain and osteoadherin, which was first located in bone. Fifty-six clones were unknown genes even though 82% matched expressed sequence tags or genomic clones. A reverse Northern dot blot showed that 96% of them were overexpressed at different rates in cultured odontoblasts. These latest results indicate that there are still unknown genes that are associated with the control of the odontoblast phenotype. Thus, cloning of odontoblast differentiation-associated genes not only opens up new methods of elucidating the normal development but also the recruitment of odontoblasts when required to initiate repair of dentin.
Assuntos
Biblioteca Gênica , Odontoblastos/metabolismo , Sequência de Bases , Diferenciação Celular/genética , Células Cultivadas , Primers do DNA/genética , DNA Complementar/genética , Expressão Gênica , Humanos , Odontoblastos/citologia , Odontogênese/genética , Reação em Cadeia da Polimerase/métodos , Proteína ReelinaRESUMO
Because the extracellular matrices of dentin and bone are composed mainly of type I collagen, their characteristics are determined by the nature of noncollagenous proteins (NCPs). Among these NCPs, some proteoglycans (PGs) belong to the small leucine-rich proteoglycans (SLRPs). Recently, osteoadherin (OSAD) has been described as a new member of this family, that is expressed by mature bovine osteoblasts. Here, we report the expression of OSAD messenger RNA (mRNA) in human dental tissues and during the development of rat molars, using in situ hybridization. For this purpose, we constructed a probe for OSAD mRNA transcripts from human odontoblast cells cultured in vitro. Our results indicate that the mature human odontoblasts overexpress the OSAD gene as compared with cells present in the pulp core. In rat developing molars, mRNA transcripts were first detected in alveolar bone in 19-day-old embryos. At the same age, no signal was detected in any cell of the first molar. In more mature teeth (newborn and 2-day-old rats), OSAD expression starts in the polarized odontoblasts and increases in the secretory and mature odontoblasts, respectively. Interestingly, a similar pattern of expression was observed in the ameloblast layer responsible for the deposition of enamel mineralized matrix. Together, these results lead us to speculate that OSAD may be implicated in biomineralization processes.