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Previous issue date: 2013-03-25 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Human mesenchymal stem cells (MSC) are powerful sources for cell therapy in regenerative medicine. The long time cultivation can result in replicative senescence or can be related to the emergence of chromosomal alterations responsible for the acquisition of tumorigenesis features in vitro. In this study, for the first time, the expression profile of MSC with a paracentric chromosomal inversion (MSC/inv) was compared to normal karyotype (MSC/n) in early and late passages. Furthermore, we compared the transcriptome of each MSC in early passages with late passages. MSC used in this study were obtained from the umbilical vein of three donors, two MSC/n and one MSC/inv. After their cryopreservation, they have been expanded in vitro until reached senescence. Total RNA was extracted using the RNeasy mini kit (Qiagen) and marked with the GeneChip ? 3 IVT Express Kit (Affymetrix Inc.). Subsequently, the fragmented aRNA was hybridized on the microarranjo Affymetrix Human Genome U133 Plus 2.0 arrays (Affymetrix Inc.). The statistical analysis of differential gene expression was performed between groups MSC by the Partek Genomic Suite software, version 6.4 (Partek Inc.). Was considered statistically significant differences in expression to p-value Bonferroni correction ˂.01. Only signals with fold change ˃ 3.0 were included in the list of differentially expressed. Differences in gene expression data obtained from microarrays were confirmed by Real Time RT-PCR. For the interpretation of biological expression data were used: IPA (Ingenuity Systems) for analysis enrichment functions, the STRING 9.0 for construction of network interactions; Cytoscape 2.8 to the network visualization and analysis bottlenecks with the aid of the GraphPad Prism 5.0 software. BiNGO Cytoscape pluggin was used to access overrepresentation of Gene Ontology categories in Biological Networks. The comparison between senescent and young at each group of MSC has shown that there is a difference in the expression parttern, being higher in the senescent MSC/inv group. The results also showed difference in expression profiles between the MSC/inv versus MSC/n, being greater when they are senescent. New networks were identified for genes related to the response of two of MSC over cultivation time. Were also identified genes that can coordinate functional categories over represented at networks, such as CXCL12, SFRP1, xvi EGF, SPP1, MMP1 e THBS1. The biological interpretation of these data suggests that the population of MSC/inv has different constitutional characteristics, related to their potential for differentiation, proliferation and response to stimuli, responsible for a distinct process of replicative senescence in MSC/inv compared to MSC/n. The genes identified in this study are candidates for biomarkers of cellular senescence in MSC, but their functional relevance in this process should be evaluated in additional in vitro and/or in vivo assays / C?lulas-tronco mesenquimais humanas (CTMH) s?o muito ?teis na terapia celular. O longo per?odo de cultivo pode resultar em senesc?ncia replicativa ou estar relacionado com o aparecimento de altera??es cromoss?micas respons?veis pela aquisi??o de um car?ter tumorig?nico in vitro . Neste estudo, foi comparado o transcriptoma de CTMH jovens e senescentes obtidas de diferentes doadores. Al?m disso, pela primeira vez, o perfil de express?o de CTMH com uma invers?o cromoss?mica parac?ntrica (CTMH/inv) foi comparado ao de CTMH que possuem cari?tipo normal (CTMH/n) em passagens jovens e senescentes de cultivo in vitro . As CTMH utilizadas neste estudo foram isoladas da veia do cord?o umbilical de tr?s dadores, dois CTMH/n e de um CTMH/inv. Ap?s a criopreserva??o, elas foram expandidas in vitro at? alcan?arem a senesc?ncia. O RNA total foi extra?do utilizando o RNeasy mini kit (Qiagen), marcado, purificado e fragmentado com o ? 3 GeneChip IVT expresso Kit (Affymetrix, Inc.). Subsequentemente, o RNA fragmentado foi hibridado no microarranjo Affymetrix Human Genome U133 Plus 2.0 (Affymetrix, Inc.). A an?lise estat?stica da express?o diferencial foi realizada usando o Partek Suite Software Genomic, vers?o 6.4 (Partek, Inc.). Foram consideradas estatisticamente significativas as diferen?as na express?o com valor de P ˂0.01 corrigido com Bonferroni. Apenas os sinais com fold change ˃3.0 foram inclu?dos na lista de diferencialmente expressos. Diferen?as na express?o g?nica observadas no estudo dos microarranjos foram confirmadas por resultados de RT-PCR em tempo real. Para a interpreta??o biol?gica dos dados foram utilizados: IPA (Ingenuity Systems) para an?lise de enriquecimento de fun??es; STRING 9,0 para a constru??o de redes de intera??es; Cytoscape 2,8 para a visualiza??o das redes e an?lises de gargalos com o aux?lio do software GraphPad Prism 5.0. O pluggin BiNGO do Cytoscape foi utilizado para avaliar a representa??o de categorias funcionais no Gene Ontology nas redes biol?gicas. A compara??o entre senescentes e jovens em cada grupo de CTMH mostrou que h? uma diferen?a no perfil de express?o, sendo maior nas senescentes do grupo CTMH/inv. Os resultados tamb?m mostraram que h? diferen?a nos perfis de express?o entre as CTMH/inv e CTMH/n, sendo maior a diferen?a quando as c?lulas est?o senescentes. Novas xiv redes foram identificadas para genes relacionados com a resposta ao longo do tempo de cultivo nos dois grupos de CTMH. Foram identificados genes que podem coordenar fun??es importantes mais enriquecidas nas redes, como por exemplo, CXCL12, SFRP1, EGF, SPP1, MMP1 e THBS1. A interpreta??o biol?gica destes dados sugere que a popula??o de c?lulas CTMH/inv tem diferentes caracter?sticas constitucionais, relacionadas com o seu potencial de prolifera??o, diferencia??o e resposta a est?mulos, respons?veis por um processo de senesc?ncia replicativa em CTMH/inv distinto das CTMH/n. Os genes identificados neste estudo s?o candidatos a marcadores da senesc?ncia celular em CTMH, mas a sua relev?ncia funcional neste processo deve ser testada em experi?ncias adicionais in vitro e/ou in vivo
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/12650 |
| Date | 25 March 2013 |
| Creators | Tavares, Joana Cristina Medeiros |
| Contributors | CPF:32398336468, http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781004Y8, Corn?lio, D?borah Afonso, CPF:78057035900, http://lattes.cnpq.br/7690257454964600, Lenz, Guido, CPF:84604131953, http://lattes.cnpq.br/4178667286777514, Ortega, Jos? Miguel, CPF:05950126807, http://lattes.cnpq.br/1919128137338097, Souza, Sandro Jos? de, CPF:70990409953, http://lattes.cnpq.br/8479967495464590, Medeiros, S?lvia Regina Batistuzzo de |
| Publisher | Universidade Federal do Rio Grande do Norte, Programa de P?s-Gradua??o em Biotecnologia, UFRN, BR, Biotecnologia Industrial; Biotecnologia em Agropecu?ria; Biotecnologia em Recursos Naturais; Biotecn |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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