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Doctoral Thesis
DOI
10.11606/T.25.2015.tde-25112015-110259
Document
Author
Full name
Ana Paula Fernandes
Institute/School/College
Knowledge Area
Date of Defense
Published
Bauru, 2015
Supervisor
Committee
Valarelli, Thais Marchini de Oliveira (President)
Borsato, Maria Cristina
Machado, Maria Aparecida de Andrade Moreira
Oliveira, Rodrigo Cardoso de
Sakai, Vivien Thiemy
Title in Portuguese
Efeitos de TGF-1 em células-tronco pulpares
Keywords in Portuguese
Células-tronco
Fator de crescimento transformador beta
Odontoblastos
Abstract in Portuguese
O objetivo deste trabalho foi avaliar, in vitro, os efeitos de diferentes concentrações do fator de crescimento transformador beta 1 (TGF-β1) em células-tronco derivadas da polpa de dentes decíduos esfoliados humanos (SHED), com relação à viabilidade, proliferação, migração e diferenciação celular. As SHED foram mantidas em meio de cultura MEMα + soro fetal bovino (FBS) 10% + penicilina e estreptomicina 1% e tratadas com TGF-β1 na concentração de 1,0; 5,0 e 10,0 ng/mL. Após 1, 3, 5 e 7 dias, foram avaliadas a viabilidade celular pelo método MTT e a proliferação pelo método SRB. Após 24 h de tratamento com TGF-β1, foi realizado um ensaio de migração celular por meio de insertos com poros de 8 μm. Para a avaliação da diferenciação celular de SHED em odontoblastos foram analisados por meio da RT-PCR os marcadores DSPP e DMP-1, após tratamento com TGF-β1 nas diferentes concentrações por 14 dias. Os resultados foram submetidos à ANOVA seguido do teste de Tukey. Em relação à viabilidade celular, as diferentes concentrações de TGF-β1 não tiveram efeito citotóxico sobre SHED. As células tratadas com diferentes concentrações de TGF-β1 apresentaram maiores taxas de proliferação que as do controle negativo (MEMα + 10% de FBS) a partir do 3o dia (p=0,000). Observou-se maiores taxas de migração em direção aos meios contendo TGF-β1, mas sem diferença estatisticamente significativa entre as diferentes concentrações utilizadas, entretanto, houve diferença estatisticamente significativa entre as diferentes concentrações de TGF-β1 com o controle positivo (p=0,000), controle negativo (p=0,000) e entre o controle positivo e negativo (p=0,002). A expressão de DMP-1 foi observada de forma crescente nas doses de 1,0 e 5,0 ng/mL de TGF-β1 ao longo do período (1, 7 e 14 dias) e na dose de 10,0 ng/mL a marcação foi mais intensa desde o primeiro dia do estímulo. Em relação à expressão de DSPP, o grupo tratado com 10,0 ng/mL apresentou marcação após 14 dias de tratamento. Sendo assim, este estudo permite concluir que as diferentes concentrações de TGF-β1 estimularam a proliferação e migração celular, sem efeito citotóxico sobre as células ao longo do período do estudo. Em relação à diferenciação celular a concentração 10,0 ng/mL de TGF-β1 estimulou à expressão de DMP-1 e DSPP.
Title in English
Keywords in English
Odontoblasts
Stem cells
Transforming growth factor beta
Abstract in English
The aim of this study was to evaluate, in vitro, the effect of transforming growth factor beta 1 (TGF-β1) in stem cells derived from the pulp of human exfoliated deciduous teeth (SHED) regarding to cell viability, proliferation, migration and differentiation. SHED were maintained in MEMα culture medium + 10% fetal bovine serum (FBS) + 1% penicillin and streptomycin, and treated with TGF-β1 at the following concentrations of 1.0; 5.0 and 10.0 ng/mL. After 1, 3, 5 and 7 days, cell viability was assessed by MTT assay and proliferation by the SRB method. After 24h of TGF-β1 treatment, cell migration assay was carried out using inserts of 8 μm pore size. To evaluate SHED differentiation into odontoblasts, DMP-1 and DSPP markers were analyzed by RT-PCR, after treatment at different concentrations of TGF-β1 for 14 days. The results were submitted by ANOVA and Tukey test. With respect to cell viability, the different TGF-β1 concentrations did not have cytotoxic effect on SHED. The cells treated by different TGF-β1 concentrations showed higher proliferation rates than those of the negative control (MEMα + 10% FBS) after the third day (p = 0.000). Higher rates of migration towards the media containing TGF-β1 were observed, but there were no statistically significant differences among the concentrations. All different TGF-β1 concentrations showed statistically significant differences with the positive control (p=0.000) and negative control (p=0.000). Statistically significant differences were observed between positive and negative control (p=0.002). DMP-1 expression was observed incrementally at TGF-β1 concentrations of 1.0 and 5.0 ng/mL at 1, 7, and 14 days and the concentration of 10.0 ng/mL was more intense from day one of the stimulus. DSPP expression was more intense after 14 days of treatment with the concentration of 10.0 ng/mL. Thus, this study concluded that different TGF-β1 concentrations stimulated cell proliferation and migration, without cytotoxic effect on the cells throughout the study period. From the perspective of cell differentiation, TGF-β1 concentration of 10.0 ng/mL was capable of stimulating DMP-1 and DSPP expression.
 
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Publishing Date
2015-11-26
 
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