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Master's Dissertation
DOI
Document
Author
Full name
Simone Ortiz Moura Fideles
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2018
Supervisor
Committee
Beloti, Márcio Mateus (President)
Martins, Manoela Domingues
Prado, Karina Fittipaldi Bombonato
Ruiz, Karina Gonzales Silvério
Title in Portuguese
Análise da expressão gênica em larga escala de células-tronco mesenquimais e de osteoblastos
Keywords in Portuguese
Células-tronco
Expressão gênica
Osteoblastos
Abstract in Portuguese
A terapia celular baseada na utilização de células-tronco mesenquimais (CTMs) tem sido considerada uma estratégia promissora para regenerar o tecido ósseo. Apesar da medula óssea constituir a principal fonte de CTMs, o tecido adiposo tem sido investigado como uma fonte alternativa, devido a maior disponibilidade e facilidade de obtenção dessas células. Porém, as CTMs obtidas de fontes distintas podem exibir diferenças a nível molecular, que ainda não estão esclarecidas pela literatura e que poderiam influenciar o potencial regenerativo dessas células. Assim sendo, o objetivo desse estudo foi comparar os perfis de expressão gênica em larga escala de CTMs isoladas da medula óssea (CTMs-MO) e do tecido adiposo (CTMs-TA) de ratos, e de osteoblastos (OBs) diferenciados a partir dessas células (OBs-MO e OBs-TA, respectivamente). As células foram cultivadas em meio de crescimento para manutenção das características de CTMs ou em meio osteogênico para indução da diferenciação osteoblástica. O RNA total das células foi extraído e a análise genômica foi realizada pela tecnologia de microarray utilizando lâminas Agilent 4x44k. Os perfis de expressão gênica foram analisados pelo software GeneSpring 14.8 GX, considerando o tecido de origem (CTMs-TA vs CTMsMO e OBs-TA vs OBs-MO) e a diferenciação celular (OBs-MO vs CTMs-MO e OBs-TA vs CTMs-TA) como parâmetros de comparação entre os grupos (fold change 2.0; p 0.05). Os dados do microarray foram confirmados por PCR em tempo real para todos os genes de interesse avaliados. Os perfis de expressão gênica entre as CTMs obtidas de fontes distintas (CTMs-TA vs CTMs-MO) e entre os OBs diferenciados a partir destas células (OBs-TA vs OBs-MO) mostraram que as células provenientes da medula óssea apresentaram uma sobreexpressão de genes relacionados à osteogênese enquanto que, as células provenientes do tecido adiposo, uma sobre-expressão de genes relacionados à angiogênese, independente da diferenciação celular. Adicionalmente, as células provenientes do tecido adiposo (CTMs e OBs) apresentaram uma sobre-expressão de genes relacionados à diferenciação adipogênica. Esses resultados, em conjunto, mostraram que as células preservaram características do tecido de origem. As análises comparativas entre as células indiferenciadas e diferenciadas obtidas da mesma fonte (OBs-MO vs CTMs-MO e OBs-TA vs CTMs-TA) mostraram que as CTMs, independente do tecido de origem, apresentaram uma sobre-expressão de genes relacionados com os processos de angiogênese, diferenciação osteoblástica e morfogênese do osso, o que sugere que as CTMs podem estar mais envolvidas com os eventos iniciais que regem o processo regenerativo que os OBs. A análise do dendrograma mostrou que os OBs-TA apresentaram um padrão de expressão gênica mais próximo ao das CTMs, em termos de similaridade e, os OBsMO, um padrão mais distante, o que sugere que os OBs-MO possam ter atingido um estágio de diferenciação celular mais avançado que os OBs-TA. Os resultados mostraram diferenças moleculares entre as populações de células que poderiam implicar em utilizações terapêuticas específicas, sugerindo que as células provenientes do tecido adiposo seriam mais indicadas para terapias angiogênicas enquanto que, as células provenientes da medula óssea, mais indicadas para terapias osteogênicas
Title in English
Large-scale gene expression analysis of mesenchymal stem cells and osteoblasts
Keywords in English
Gene expression
Osteoblasts
Stem cells
Abstract in English
Cell therapy based on the use of mesenchymal stem cells (MSCs) has been considered a promising strategy to regenerate bone tissue. Although bone marrow represents the main source of MSCs, adipose tissue has been investigated as an alternative source due to the greater availability and ease of isolation of these cells. However, MSCs obtained from different sources may show differences at the molecular level, which are not yet established in the literature and that could influence the regenerative potential of these cells. Thus, the aim of this study was to compare the large-scale gene expression profiles of MSCs isolated from bone marrow (BMMSCs) and adipose tissue (AT-MSCs) of rats, and of the osteoblasts differentiated from these cells (BM-OBs and AT-OBs, respectively). Cells were cultured in growth medium to maintain the characteristics of MSCs or in osteogenic medium to induction of osteoblastic differentiation. Total RNA was extracted and genomic analysis was performed by microarray technology using Agilent 4x44k slides. The gene expression profiles were analyzed by GeneSpring 14.8 GX software, considering the source (AT-MSCs vs BM-MSCs and AT-OBs vs BM-OBs) and cell differentiation (BM-OBs vs BM-MSCs and AT-OBs vs AT-MSCs) as parameters of comparison between the groups (fold change 2.0; p 0.05). The microarray data were confirmed by real-time PCR for all the interest genes evaluated. The gene expression profiles between the MSCs obtained from different sources (AT-MSCs vs BM-MSCs) and between the OBs differentiated from these cells (AT-OBs vs BM-OBs) showed that the cells from the bone marrow presented an overexpression of genes related to osteogenesis whereas the cells from adipose tissue showed an overexpression of genes related to angiogenesis, independent of cell differentiation. In addition, cells from adipose tissue (MSCs and OBs) showed an overexpression of genes related to adipogenic differentiation. These results, together, showed that the cells preserved characteristics of the source. Comparative analyses between the undifferentiated and differentiated cells obtained from the same source (BM-OBs vs BM-MSCs and AT-OBs vs AT-MSCs) showed that MSCs, regardless of source, showed an overexpression of genes related to the angiogenesis, osteoblastic differentiation and bone morphogenesis process, suggesting that MSCs may be more involved with the early events that govern the regenerative process than OBs. Dendrogram analysis showed that AT-OBs had a gene expression profile closer to MSCs in terms of similarity and the BM-OBs had a more distant profile, suggesting that BM-OBs may have reached a stage of cell differentiation more advanced than AT-OBs. The results showed molecular differences between cell populations that could imply in specific therapeutic uses, suggesting that cells derived from adipose tissue would be better suited for angiogenic therapies, whereas bone marrow cells are more suitable for osteogenic therapies
 
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Publishing Date
2019-07-17
 
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