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Master's Dissertation
DOI
https://doi.org/10.11606/D.23.2014.tde-12082014-183505
Document
Author
Full name
Luiz Henrique Santos Silva
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2014
Supervisor
Committee
Paiva, Katiucia Batista da Silva (President)
Braga, Patricia Cristina Baleeiro Beltrao
Pobocik, Andrea Mantesso
Title in Portuguese
Expressão gênica de moléculas da matriz extracelular e da membrana celular durante a diferenciação de células-tronco adultas da polpa dentária humana
Keywords in Portuguese
Célula-tronco da polpa dentária
Diferenciação Osteogênica
Inibidores das Metaloproteinases de matriz
Metaloproteinases de matriz
Proteínas da membrana celular
Abstract in Portuguese
As células-tronco mesenquimais (MSCs) são células multipotentes que tem o potencial de se diferenciarem em várias linhagens celulares in vitro e in vivo. Estas são encontradas em nichos específicos em muitos órgãos e tecidos adultos, tais como medula óssea, tecido adiposo, músculo, dente, cordão umbilical, pele, cartilagem articular, sendo facilmente isoladas, expandidas e com alta capacidade proliferativa in vitro. Assim, estas características têm despertado grande interesse na sua utilização como uma potencial fonte de células para o reparo e regeneração tecidual de diversos órgãos e tecidos. Pouco se conhece sobre as moléculas que são secretadas pelas MSCs para a matriz extracelular (MEC) e que estão na interface célula-matriz e estão presentes em vias de transdução de sinais intracelulares. Desta forma, o objetivo deste trabalho foi avaliar o perfil de expressão gênica de enzimas que remodelam a MEC (metaloproteinases de matriz MMPs: 15 membros) e seus inibidores (inibidores teciduais das metaloproteinases de matriz TIMPs: 4 membros e RECK) e proteína da membrana plasmática (Caveolina-1) durante a diferenciação osteogenica in vitro a partir de células-tronco mesenquimais da polpa dentária humana (DPSCs). Para tanto, utilizamos polpas dentárias humanas provenientes de terceiros molares de indivíduos adultos (18-32 anos n=3) e as DPSCs isoladas foram imunofenotipadas por citometria de fluxo, avaliada a taxa de proliferação, induzidas as diferenciações osteogênica (1, 7, 14, 21 e 28 dias) e adipogênica (28 dias) e os transcritos avaliados por PCR em tempo real. Estas células foram positivas para o marcadores CD29, CD105, STRO-1, CD44, CD90 negativas os marcadores para CD31, CD45, CD34 e CD14 e são capazes de se diferenciarem em osteoblastos e adipócitos. Verificamos que as MMP-2, MMP-3, MMP-13, MMP-14, MMP-25, TIMP-3, TIMP-4 e Caveolina-1 foram diferencialmente expressas durante a diferenciação osteogênica, sendo reguladas positivamente apenas no período de 28 dias pós indução e a TIMP-1 regulada positivamente desde o primeiro dia de indução. A MMP-11 e MMP-16 não foram detectadas nas DPSCs e nem durante a diferenciação osteogênica. Desta forma, concluímos que MMPs encontradas bem como a Caveolina-1 e as TIMP-3 e TIMP-4 podem estar participando dos dos eventos de diferenciação óssea em DPSCs, a TIMP-1 pode estar participando de eventos biológicos relacionados as propriedades do estado indiferenciado das DPSCs e da diferenciação óssea e que as MMP-11 e MMP-16 não são expressas pelas DPSCs e também não estão envolvidas na diferenciação osteogênica.
Title in English
Gene expression of extracellular matrix and cell membrane molecules during cellular differentiation from human dental pulp stem cells
Keywords in English
Cell membrane proteins
Dental pulp stem cells
Inhibitors of matrix metalloproteinases
Matrix metalloproteinases
Oteogenic differentiation
Abstract in English
Mesenchymal stem cells (MSCs) are multipotent cells that have the potential to differentiate into various cell lineages in vitro and in vivo. These are found in specific niches in many adult organs and tissues, such as bone marrow, adipose tissue, muscle, tooth, umbilical cord, skin, cartilage, being easily isolated, expanded and high proliferative capacity in vitro. Thereby, these features have attracted great interest in its use as a potential source of cells for tissue repair and regeneration of various organs and tissues. Little is known about the molecules secreted by MSCs into the extracellular matrix (ECM), present at cell-matrix interface and present on intracellular signal transduction. Thus, the aim of this study was to evaluate gene expression profile of ECM remodeling enzymes (matrix metalloproteinases MMPs: 15 members) and their inhibitors (tissue inhibitors of matrix metalloproteinases TIMPs: 4 members and RECK) and plasma membrane proteins (Caveolin-1) that participate in signaling pathways during osteogenic differentiation in vitro from human dental pulp stem cells (DPSCs). Normal human impacted third molars were collected from adults (18-32 years-old n=3) and DPSCs isolated were immunophenotyping by flow cytometry, evaluated the proliferation ratio, induced to osteogenic (1, 7, 14, 21 and 28-days) and adipogenic differentiation (28-days) and the transcript levels evaluated by Real Time PCR. These cells are positive for CD29, CD105, STRO -1, CD44, and CD90 markers and negative for CD31, CD45, CD34, and CD14 markers and are capable of differentiating into osteoblasts and adipocytes. We found that MMP- 2, MMP -3, MMP -13, MMP -14, MMP -25, TIMP-3, TIMP-4 and Caveolin-1 were differentially expressed during osteogenic differentiation, being upregulated only at 28 days post-induction and TIMP-1 upregulated from the first day of induction. MMP-11 and MMP-16 were not detected in DPSCs neither during differentiation. Thus, we conclude that MMPs, Caveolin-1 found as well as TIMP-3 and TIMP-4 may be participating in the event of bone differentiation in DPSCs, TIMP-1 may participate in biological events related to the properties of the undifferentiated state DPSCs and osteogenic differentiation, MMP-11 and MMP-16 are not also expressed by DPSCs and are not involved in osteogenic differentiation.
 
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Publishing Date
2014-09-26
 
WARNING: The material described below relates to works resulting from this thesis or dissertation. The contents of these works are the author's responsibility.
  • PAIVA, KATIUCIA, SILVA, LUIZ, e SOGAYAR, MARI. Differential gene expression of matrix metalloproteinases (MMPs), MMP inhibitors (TIMPs and RECK), and MMP-activator (EMMPRIN/CD147) during osteogenic differentiation from human dental pulp stem cells [doi:10.1530/boneabs.1.PP172]. Bone Abstracts [online], 2013, vol. 1, p. 99-100.
  • PAIVA, K. B. S., et al. Flow cytometric characterization of culture expanded human dental pulp stem cells (DPSCs) - expression of Caveolin-1, FAK and CD147 (EMMPRIN). In EMBO Conferences - Cellular Signalling and Molecular Medicine, Dubrovnik/Croácia, 2012. EMBO Conferences - 3rd Cellular Signalling and Molecular Medicine., 2012. Abstract.
  • SILVA, L. H. S., et al. Immuphenotyping of culture expanded human Dental Pulp Stem Cells (DPSCs): expression of Caveolin-1, FAK and CD147 (EMMPRIN). In 7. Congresso Brasileiro de Células-Tronco e Terapia Celular, São Paulo, 2012. 7. Congresso Brasileiro de Células-Tronco e Terapia Celular., 2012. Abstract.
  • SILVA, L. H. S., Sogayar, M.C., e Paiva, Katiúcia Batista da Silva. Potencial de Diferenciação Celular de Células-Tronco da Polpa Dentária Humana. In XX Reunião de Pesquisa e do XVII Seminário de Iniciação Científica da FOUSP de 2013, São Paulo, 2013. XX Reunião de Pesquisa e do XVII Seminário de Iniciação Científica da FOUSP de 2013., 2013. Resumo.
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