Isolamento, cultivo e caracterização de células derivadas de cardioesferas de camundongos CD1
Autor(a) principal: | |
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Data de Publicação: | 2013 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFU |
Texto Completo: | https://repositorio.ufu.br/handle/123456789/12764 https://doi.org/10.14393/ufu.di.2013.130 |
Resumo: | In Brazil, as in the world, cardiovascular diseases have been a major cause of death. The high mortality and few therapeutic alternatives for this disease have stimulated research on the stem cell field. Recently some groups have shown the presence of resident stem cells in the heart that can generate new cardiomyocytes. However, the cardiac stem cells exist in small amounts in vivo and their isolation and ex vivo expansion is a challenge for clinical use. In this study, our goal was to isolate, characterize and grow cardiosphere-derived cells from CD1 mice. Surface molecules present in these cells were analyzed by flow cytometry and expression of cytoplasmic proteins was analyzed by immunofluorescence. Primary cells were isolated from the hearts of adult mice after digestion of the organ into small pieces using 420U/ml of collagenase II in four cycles of 5 minutes at 37°C. Subsequently, cells were maintained in culture in an incubator at 37°C and 5% CO2 for approximately 3 weeks. The immunofluorescence assays and flow cytometry were performed between passages 3 and 5 which 0.25% trypsin was used. The cells expressed the following proteins in their cytoplasm: vimentin, desmin, nestin and α-smooth muscle actin and population doubling time was 1.8 days. Additionally, flow cytometry showed low expression of the molecules CD19 (0.4), CD45 (0.5) and CD90 (4.77), and higher expression of CD73 (71.47), CD105 (25.1), CD14 (25.17). These results suggest that the CDC isolated from CD1 mice are cells with a mesenchymal phenotype, but their beneficial potential in cardiac repair remains to be tested. |
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Isolamento, cultivo e caracterização de células derivadas de cardioesferas de camundongos CD1Isolation, culture and characterization of cardiospheres-derived cells from CD1 miceCélulas derivadas de cardioesferasCélulas tronco cardíacasCaracterizaçãoCDCsCardiac stem cellsCharacterizationCNPQ::CIENCIAS DA SAUDEIn Brazil, as in the world, cardiovascular diseases have been a major cause of death. The high mortality and few therapeutic alternatives for this disease have stimulated research on the stem cell field. Recently some groups have shown the presence of resident stem cells in the heart that can generate new cardiomyocytes. However, the cardiac stem cells exist in small amounts in vivo and their isolation and ex vivo expansion is a challenge for clinical use. In this study, our goal was to isolate, characterize and grow cardiosphere-derived cells from CD1 mice. Surface molecules present in these cells were analyzed by flow cytometry and expression of cytoplasmic proteins was analyzed by immunofluorescence. Primary cells were isolated from the hearts of adult mice after digestion of the organ into small pieces using 420U/ml of collagenase II in four cycles of 5 minutes at 37°C. Subsequently, cells were maintained in culture in an incubator at 37°C and 5% CO2 for approximately 3 weeks. The immunofluorescence assays and flow cytometry were performed between passages 3 and 5 which 0.25% trypsin was used. The cells expressed the following proteins in their cytoplasm: vimentin, desmin, nestin and α-smooth muscle actin and population doubling time was 1.8 days. Additionally, flow cytometry showed low expression of the molecules CD19 (0.4), CD45 (0.5) and CD90 (4.77), and higher expression of CD73 (71.47), CD105 (25.1), CD14 (25.17). These results suggest that the CDC isolated from CD1 mice are cells with a mesenchymal phenotype, but their beneficial potential in cardiac repair remains to be tested.Conselho Nacional de Desenvolvimento Científico e TecnológicoMestre em Ciências da SaúdeNo Brasil, como em todo o mundo, as doenças cardiovasculares têm sido uma das principais causas de morte. A alta mortalidade e as poucas alternativas terapêuticas para estas doenças têm estimulado a investigação no campo das células estaminais. Recentemente, alguns grupos têm mostrado a presença de células-tronco/progenitoras residentes no coração. Estas poderiam ser cultivadas diretamente a partir de tecidos cardíacos produzindo aglomerados esféricos denominados Cardioesferas as quais, contém células proliferativas que dão origem, após o plaqueamento, a uma população heterogênea denominada: células derivadas de cardioesferas (CDCs). O objetivo deste estudo foi isolar, cultivar e caracterizar as CDCs de camundongos da linhagem CD1. Para isto, as células primárias foram isoladas a partir de corações de camundongos adultos da linhagem CD1 após a digestão de pequenos fragmentos do órgão em colagenase tipo II 420U/ml por 20 minutos a 37°C. Nas análises por citometria de fluxo (FACS) foram observadas baixa expressão das moléculas de CD19 (0,4%), CD45 (0,5%) e CD90 (4,7%), e alta expressão das moléculas CD73 (71,4%), CD105 (25,1%), CD14 (25,1%). Nos ensaios de imunofluorescência foi possível observar a expressão das proteínas no citoplasma dos cardiomiócitos: vimentina, desmina e alfa actina de músculo liso, além da expressão do filamento intermediário nestina. Ao analisar a expansão celular por population doubling time foi observado que as CDCs duplicaram sua população original em cerca de 1,8 dias. Estes resultados sugerem que as CDCs isoladas a partir de camundongos da linhagem CD1, são células que apresentam características de células mesenquimais, constituindo uma população celular a ser testada nos estudos em terapias celulares. Estes resultados motivam o estabelecimento de protocolos que visem investigar possíveis efeitos parácrinos benéficos, bem como o potencial angiogênico e cardiogênico destas células.Universidade Federal de UberlândiaBRPrograma de Pós-graduação em Ciências da SaúdeCiências da SaúdeUFUCarvalho, Adriana Bastoshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4739933D8Resende, Elmiro Santoshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4793244Y5Beletti, Marcelo Emíliohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4703947D5Mineo, Jose Robertohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780327P9Oliveira, Patrícia Fidelis dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4700425T6Alves, Poliana Rodrigues2016-06-22T18:33:11Z2013-07-242016-06-22T18:33:11Z2013-02-28info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfapplication/pdfALVES, Poliana Rodrigues. Isolation, culture and characterization of cardiospheres-derived cells from CD1 mice. 2013. 48 f. Dissertação (Mestrado em Ciências da Saúde) - Universidade Federal de Uberlândia, Uberlândia, 2013. DOI https://doi.org/10.14393/ufu.di.2013.130https://repositorio.ufu.br/handle/123456789/12764https://doi.org/10.14393/ufu.di.2013.130porinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFUinstname:Universidade Federal de Uberlândia (UFU)instacron:UFU2022-09-05T18:50:12Zoai:repositorio.ufu.br:123456789/12764Repositório InstitucionalONGhttp://repositorio.ufu.br/oai/requestdiinf@dirbi.ufu.bropendoar:2022-09-05T18:50:12Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU)false |
dc.title.none.fl_str_mv |
Isolamento, cultivo e caracterização de células derivadas de cardioesferas de camundongos CD1 Isolation, culture and characterization of cardiospheres-derived cells from CD1 mice |
title |
Isolamento, cultivo e caracterização de células derivadas de cardioesferas de camundongos CD1 |
spellingShingle |
Isolamento, cultivo e caracterização de células derivadas de cardioesferas de camundongos CD1 Alves, Poliana Rodrigues Células derivadas de cardioesferas Células tronco cardíacas Caracterização CDCs Cardiac stem cells Characterization CNPQ::CIENCIAS DA SAUDE |
title_short |
Isolamento, cultivo e caracterização de células derivadas de cardioesferas de camundongos CD1 |
title_full |
Isolamento, cultivo e caracterização de células derivadas de cardioesferas de camundongos CD1 |
title_fullStr |
Isolamento, cultivo e caracterização de células derivadas de cardioesferas de camundongos CD1 |
title_full_unstemmed |
Isolamento, cultivo e caracterização de células derivadas de cardioesferas de camundongos CD1 |
title_sort |
Isolamento, cultivo e caracterização de células derivadas de cardioesferas de camundongos CD1 |
author |
Alves, Poliana Rodrigues |
author_facet |
Alves, Poliana Rodrigues |
author_role |
author |
dc.contributor.none.fl_str_mv |
Carvalho, Adriana Bastos http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4739933D8 Resende, Elmiro Santos http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4793244Y5 Beletti, Marcelo Emílio http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4703947D5 Mineo, Jose Roberto http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780327P9 Oliveira, Patrícia Fidelis de http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4700425T6 |
dc.contributor.author.fl_str_mv |
Alves, Poliana Rodrigues |
dc.subject.por.fl_str_mv |
Células derivadas de cardioesferas Células tronco cardíacas Caracterização CDCs Cardiac stem cells Characterization CNPQ::CIENCIAS DA SAUDE |
topic |
Células derivadas de cardioesferas Células tronco cardíacas Caracterização CDCs Cardiac stem cells Characterization CNPQ::CIENCIAS DA SAUDE |
description |
In Brazil, as in the world, cardiovascular diseases have been a major cause of death. The high mortality and few therapeutic alternatives for this disease have stimulated research on the stem cell field. Recently some groups have shown the presence of resident stem cells in the heart that can generate new cardiomyocytes. However, the cardiac stem cells exist in small amounts in vivo and their isolation and ex vivo expansion is a challenge for clinical use. In this study, our goal was to isolate, characterize and grow cardiosphere-derived cells from CD1 mice. Surface molecules present in these cells were analyzed by flow cytometry and expression of cytoplasmic proteins was analyzed by immunofluorescence. Primary cells were isolated from the hearts of adult mice after digestion of the organ into small pieces using 420U/ml of collagenase II in four cycles of 5 minutes at 37°C. Subsequently, cells were maintained in culture in an incubator at 37°C and 5% CO2 for approximately 3 weeks. The immunofluorescence assays and flow cytometry were performed between passages 3 and 5 which 0.25% trypsin was used. The cells expressed the following proteins in their cytoplasm: vimentin, desmin, nestin and α-smooth muscle actin and population doubling time was 1.8 days. Additionally, flow cytometry showed low expression of the molecules CD19 (0.4), CD45 (0.5) and CD90 (4.77), and higher expression of CD73 (71.47), CD105 (25.1), CD14 (25.17). These results suggest that the CDC isolated from CD1 mice are cells with a mesenchymal phenotype, but their beneficial potential in cardiac repair remains to be tested. |
publishDate |
2013 |
dc.date.none.fl_str_mv |
2013-07-24 2013-02-28 2016-06-22T18:33:11Z 2016-06-22T18:33:11Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
ALVES, Poliana Rodrigues. Isolation, culture and characterization of cardiospheres-derived cells from CD1 mice. 2013. 48 f. Dissertação (Mestrado em Ciências da Saúde) - Universidade Federal de Uberlândia, Uberlândia, 2013. DOI https://doi.org/10.14393/ufu.di.2013.130 https://repositorio.ufu.br/handle/123456789/12764 https://doi.org/10.14393/ufu.di.2013.130 |
identifier_str_mv |
ALVES, Poliana Rodrigues. Isolation, culture and characterization of cardiospheres-derived cells from CD1 mice. 2013. 48 f. Dissertação (Mestrado em Ciências da Saúde) - Universidade Federal de Uberlândia, Uberlândia, 2013. DOI https://doi.org/10.14393/ufu.di.2013.130 |
url |
https://repositorio.ufu.br/handle/123456789/12764 https://doi.org/10.14393/ufu.di.2013.130 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Uberlândia BR Programa de Pós-graduação em Ciências da Saúde Ciências da Saúde UFU |
publisher.none.fl_str_mv |
Universidade Federal de Uberlândia BR Programa de Pós-graduação em Ciências da Saúde Ciências da Saúde UFU |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UFU instname:Universidade Federal de Uberlândia (UFU) instacron:UFU |
instname_str |
Universidade Federal de Uberlândia (UFU) |
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UFU |
institution |
UFU |
reponame_str |
Repositório Institucional da UFU |
collection |
Repositório Institucional da UFU |
repository.name.fl_str_mv |
Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU) |
repository.mail.fl_str_mv |
diinf@dirbi.ufu.br |
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1805569645899415552 |