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Aspectos moleculares do efeito do fator de transformação de crescimento-beta1 (TGF-β1) nas vias de sinalização na biomineralização in vitro. / Molecular aspects of the effect of transforming growth factor-beta 1 (TGF-β1) in the signaling pathways in vitro biomineralization.Donato, Tatiani Ayako Goto 11 March 2014 (has links)
Este estudo in vitro teve como objetivo avaliar os efeitos moleculares do TGF-β1, com diferentes períodos de suplementação, sobre a formação do fenótipo osteogênico das células MC3T3-E1, comparando-os com células tratadas com AA+β-GP suplementados com Dex e/ou TGF-β1, sem e com a neutralização dos receptores de TGF-β1. A expressão gênica do próprio TGF-β1 e Smad3 foram analisadas, bem como, a diferenciação das células osteogênicas e a biomineralização. As células tratadas com TGF-β1 sem neutralização de receptores apresentam efeito inibitório nos estágios mais avançados da diferenciação dos osteoblastos e da biomineralização in vitro, mas expressarem alguns marcadores importantes envolvidos na mineralização. Observaram-se nódulos de mineral em todos os tratamentos das células que tiveram os receptores de TGF-β1 neutralizados, mas houve uma diminuição na expressão de alguns genes. Os resultados confirmam a complexidade da via de sinalização do TGF-β1, mostrando que existem lacunas para que seja entendido o mecanismo dessa molécula na biologia osteoblástica. / This in vitro study aimed to evaluate the molecular effects of TGF-β1, with different supplementation time periods on the establishment of MC3T3-E1 cells, comparing with cells treated with AA+β-GP supplemented with Dex and/or TGF-β1, without or with neutralization of TGF-β1 receptors. The gene expression of the TGF-β1 and Smad3 were analyzed, as well as the osteoblast differentiation and biomineralization. The cells treated with TGF-β1 without neutralization of receptors have had inhibitory effect on some important stages of osteoblast differentiation and biomineralization in vitro, but expressed some important mineralization markers. Mineral nodules were observed in all treatments of cells with their TGF-β1 receptors neutralized, but there was a decrease in the expression of some important genes. The results confirm the complexity of the pathway signaling of TGF-β1, showing that there are gaps for understand the mechanisms of this molecule in the biology of osteoblasts.
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ASARM et biominéralisation de progéniteurs pulpaires / ASARM and dental pulp stem cells mineralizationSalmon, Benjamin 02 October 2012 (has links)
Dans le rachitisme hypophosphatémique lié à l’X (XLH), MEPE (Matrix Extracellular PhosphoglycoprotEin), une protéine non collagénique impliquée dans la biominéralisation, subit un clivage pathologique de son extrémité C-terminale. Les peptides ainsi libérés sont porteurs d’un domaine ASARM (acidic serine- and aspartate- rich motif) très conservé dans l’évolution. ASARM inhibe la réabsorption tubulaire du phosphate et la minéralisation de la matrice extracellulaire osseuse. Précédemment, notre équipe a identifié des taux élevés de ce peptide ASARM dérivé de MEPE dans la dentine issue de patients XLH. Ce travail a pour objectif principal d’étudier l’effet d’ASARM sur la minéralisation dentinaire afin de mieux comprendre son implication dans les anomalies dentaires observées chez les malades. Des lattis de collagène ensemencés avec des cellules souches pulpaires SHEDs (Dental pulp stem cells derived from deciduous teeth) englobés dans une tranche de dent humaine ont été cultivés dans des conditions d’induction odontoblastique avec et sans 20 µM de chacune des formes phosphorylé (p-ASARM) ou non phosphorylé (np-ASARM) du peptide recombinant. La minéralisation a été appréciée par microscopie électronique à balayage et colorations de von Kossa. L’expression des marqueurs odontogéniques (DSPP, ostéocalcine, MEPE) a été évaluée par immunohistochimie, qPCR et Western-blot. Parallèlement, des billes d’agarose imprégnées p-ASARM et np-ASARM ont été implantées dans un modèle d’effraction pulpaire chez le rat, dans lequel un pont de dentine de réparation se forme spontanément. La minéralisation dans la chambre pulpaire a été évaluée par micro-CT et immunohistochimie. Dans le modèle in vitro 3D, p-ASARM a inhibé la différenciation des SHEDs, ce qui s’est traduit par 1) l’absence de formation de nodule de minéralisation, 2) la diminution des marqueurs odontogéniques, 3) la surexpression de MEPE, comparativement au contrôle ou au traitement du milieu par np-ASARM. In vivo, p-ASARM a perturbé le processus de réparation dentinaire et a entrainé une surexpression de MEPE. Ces résultats confirment notre hypothèse selon laquelle p-ASARM inhibe la différenciation odonblastique et la minéralisation de la dentine. De plus, l’effet inducteur de p-ASARM sur l’expression de MEPE suggère l’existence d’une boucle de rétrocontrôle positif impliquée dans l’étiopathogénie du XLH. Ainsi, les défauts de minéralisation de la dentine hypophosphatémique sont probablement une conséquence de la libération du peptide ASARM dans la matrice extracellulaire. / In X-linked familial hypophosphatemic rickets (XLH), MEPE (Matrix Extracellular PhosphoglycoprotEin) is cleaved, releasing phosphorylated ASARM (acidic serine- and aspartate- rich motif) peptides that inhibit mineralization of bone extracellular matrix (ECM), and renal tubular phosphate reabsorption. We recently identified high levels of MEPE-derived ASARM peptides in human XLH dentin. The present study was aimed to investigate their effects on dentin mineralization in order to better understand their role in the etiology of tooth abnormalities observed in XLH patients. Dental pulp stem cells derived from deciduous teeth (SHEDs) were seeded in a collagen scaffold, cultured in human tooth slices under mineralizing conditions as a control, and with 20 µM of either phosphorylated (p-ASARM) or non-phosphorylated (np-ASARM) MEPE-derived ASARM peptides. Mineralization was assessed by scanning electron microscopy and von Kossa staining. Odontogenic markers (DSPP, osteocalcin, MEPE) were assessed by immunohistochemistry, RT-PCR and Western blot. In parallel, agarose beads soaked with recombinant ASARM peptides were implanted in a rat pulp injury model where a reparative dentin bridge is spontaneously formed; the repair process was evaluated by micro-CT and IHC. In the tooth slice culture model, p-ASARM inhibited SHED differentiation, with 1) no formation of mineralization nodule, 2) decreased odontogenic marker expression, and 3) up-regulation of MEPE expression, in contrast with np-ASARM and control. In the rat pulp injury model, p-ASARM impaired the formation of the reparative dentin bridge and increased MEPE expression. The present data support our hypothesis that p-ASARM impairs odontogenic differentiation process and the resulting mineralization of dentin. Moreover, the identification of a stimulating effect of p-ASARM on MEPE expression suggests a positive feedback loop in the pathogenicity of XLH disease. Accordingly, the mineralized defects in XLH tooth dentin may be a direct consequence of the release of ASARM peptides in the ECM.
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Aspectos moleculares do efeito do fator de transformação de crescimento-beta1 (TGF-β1) nas vias de sinalização na biomineralização in vitro. / Molecular aspects of the effect of transforming growth factor-beta 1 (TGF-β1) in the signaling pathways in vitro biomineralization.Tatiani Ayako Goto Donato 11 March 2014 (has links)
Este estudo in vitro teve como objetivo avaliar os efeitos moleculares do TGF-β1, com diferentes períodos de suplementação, sobre a formação do fenótipo osteogênico das células MC3T3-E1, comparando-os com células tratadas com AA+β-GP suplementados com Dex e/ou TGF-β1, sem e com a neutralização dos receptores de TGF-β1. A expressão gênica do próprio TGF-β1 e Smad3 foram analisadas, bem como, a diferenciação das células osteogênicas e a biomineralização. As células tratadas com TGF-β1 sem neutralização de receptores apresentam efeito inibitório nos estágios mais avançados da diferenciação dos osteoblastos e da biomineralização in vitro, mas expressarem alguns marcadores importantes envolvidos na mineralização. Observaram-se nódulos de mineral em todos os tratamentos das células que tiveram os receptores de TGF-β1 neutralizados, mas houve uma diminuição na expressão de alguns genes. Os resultados confirmam a complexidade da via de sinalização do TGF-β1, mostrando que existem lacunas para que seja entendido o mecanismo dessa molécula na biologia osteoblástica. / This in vitro study aimed to evaluate the molecular effects of TGF-β1, with different supplementation time periods on the establishment of MC3T3-E1 cells, comparing with cells treated with AA+β-GP supplemented with Dex and/or TGF-β1, without or with neutralization of TGF-β1 receptors. The gene expression of the TGF-β1 and Smad3 were analyzed, as well as the osteoblast differentiation and biomineralization. The cells treated with TGF-β1 without neutralization of receptors have had inhibitory effect on some important stages of osteoblast differentiation and biomineralization in vitro, but expressed some important mineralization markers. Mineral nodules were observed in all treatments of cells with their TGF-β1 receptors neutralized, but there was a decrease in the expression of some important genes. The results confirm the complexity of the pathway signaling of TGF-β1, showing that there are gaps for understand the mechanisms of this molecule in the biology of osteoblasts.
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