Caracterização do estado indiferenciado de células tronco embrionárias murinas expandidas na presença de nanopartículas magnéticas e isolamento de células tronco embrionárias a partir de blastócitos bovinos
Autor(a) principal: | |
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Data de Publicação: | 2009 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFG |
Texto Completo: | http://repositorio.bc.ufg.br/tede/handle/tde/1014 |
Resumo: | Magnetic nanoparticles (MNPs) have been used in a great variety of biomedical applications, especially in cancer treatment, drug delivery, and diagnosis by magnetic resonance imaging. Embryonic stem cells (ESCs), due to its capacity of auto-renewal and differentiation in some types of cells, offer a great potential for its use in tissue regeneration and in alternative treatments for many degenerative diseases. The study had as aims: i) to evaluate in vitro citotoxicity of maghemite nanoparticles functionalized with lauric acid, DMSA, and citrate in human melanoma cells (SK-MEL-37) by the MTT assay, electronic microscopy, and DNA electroforesis by agarose gel; ii) to develop a culture system using the previously selected MNPs and a magnet to expand in vitro murine embryonic stem cells (mESCs) in the absence of a co-culture of murine embryonic fibroblasts (MEF) (the indifferentiated state of the mES was analyzed by alkaline phosphatase cytochemistry, electronic microscopy, and analysis of Oct-4 and Nanog gene expression by RT-PCR); iii) to isolate and expand ESCs from bovine blastocysts, and to characterize its pluripotency by analysis of Oct-4 and STAT-3 gene expression by RT-PCR. The MNPs coated with lauric acid, citrate, and DMSA showed no citotoxicity, judging by the high values of IC50 found (254, 433 and 2260 μg-iron/ml, respectively), and that the nanoparticles coated with citrate was chosen to expand the mESCs. The doubling time for the cells cultivated in the presence of MNPs was slightly higher than in the presence of MEF (20.67 ± 0.19 vs. 15.95 ± 0.21) (p= 0.001). The mESCs cultivated in the presence of MNPs showed morphology similar to ESCs, and its pluripotency was confirmed by expression of indifferentiation markers Oct-4 and Nanog by RT-PCR and high alkaline phosphatase activity. One bovine embryonic stem cell (bESCs) line was obtained and maintained by six subcultures for 60 days period. The pluripotency of the bESCs was confirmed morphologically as well as by Oct-4 e STAT-3 gene expression |
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GUILLO, Lídia Andreuhttp://lattes.cnpq.br/3401436781775091http://lattes.cnpq.br/5011536994328123FREITAS, Erika Regina Leal de2014-07-29T15:10:32Z2010-02-052009-08-14FREITAS, Erika Regina Leal de. Characterization of undifferentiated state of murine embryonic stem cells expanded in the presence of magnetic nanoparticles and isolation of embryonic stem cells from bovine blastocysts. 2009. 127 f. Tese (Doutorado em Ciencias Biologicas) - Universidade Federal de Goiás, Goiânia, 2009.http://repositorio.bc.ufg.br/tede/handle/tde/1014Magnetic nanoparticles (MNPs) have been used in a great variety of biomedical applications, especially in cancer treatment, drug delivery, and diagnosis by magnetic resonance imaging. Embryonic stem cells (ESCs), due to its capacity of auto-renewal and differentiation in some types of cells, offer a great potential for its use in tissue regeneration and in alternative treatments for many degenerative diseases. The study had as aims: i) to evaluate in vitro citotoxicity of maghemite nanoparticles functionalized with lauric acid, DMSA, and citrate in human melanoma cells (SK-MEL-37) by the MTT assay, electronic microscopy, and DNA electroforesis by agarose gel; ii) to develop a culture system using the previously selected MNPs and a magnet to expand in vitro murine embryonic stem cells (mESCs) in the absence of a co-culture of murine embryonic fibroblasts (MEF) (the indifferentiated state of the mES was analyzed by alkaline phosphatase cytochemistry, electronic microscopy, and analysis of Oct-4 and Nanog gene expression by RT-PCR); iii) to isolate and expand ESCs from bovine blastocysts, and to characterize its pluripotency by analysis of Oct-4 and STAT-3 gene expression by RT-PCR. The MNPs coated with lauric acid, citrate, and DMSA showed no citotoxicity, judging by the high values of IC50 found (254, 433 and 2260 μg-iron/ml, respectively), and that the nanoparticles coated with citrate was chosen to expand the mESCs. The doubling time for the cells cultivated in the presence of MNPs was slightly higher than in the presence of MEF (20.67 ± 0.19 vs. 15.95 ± 0.21) (p= 0.001). The mESCs cultivated in the presence of MNPs showed morphology similar to ESCs, and its pluripotency was confirmed by expression of indifferentiation markers Oct-4 and Nanog by RT-PCR and high alkaline phosphatase activity. One bovine embryonic stem cell (bESCs) line was obtained and maintained by six subcultures for 60 days period. The pluripotency of the bESCs was confirmed morphologically as well as by Oct-4 e STAT-3 gene expressionAs nanopartículas magnéticas (NPM) têm sido utilizadas em inúmeras aplicações biomédicas, destacando-se no tratamento do câncer, carreamento de drogas e diagnóstico por imagem de ressonância. As células tronco embrionárias (ES), devido à sua capacidade de auto-renovação e de diferenciação em vários tipos de células, oferecem um grande potencial para sua utilização na regeneração de tecidos e em tratamentos alternativos para muitas doenças degenerativas. Os objetivos do estudo foram: i) avaliar a citotoxicidade in vitro de NPM de maghemita funcionalizadas com ácido láurico, DMSA e citrato em células de melanoma humano (SK-MEL-37) através de ensaio de MTT, microscopia eletrônica e eletroforese de DNA em gel de agarose; ii) desenvolver um sistema de cultura utilizando as NPM previamente selecionadas e um magneto para expandir in vitro células tronco embrionárias murinas (mES) na ausência de co-cultura de fibroblastos embrionários murinos (MEF) (o estado indiferenciado das células mES foi analisado por citoquímica para fosfatase alcalina, por microscopia eletrônica e análise da expressão de genes Oct-4 e Nanog por RT-PCR); iii) isolar e expandir células ES a partir de blastocistos bovinos, e caracterizar a sua pluripotência por meio da análise da expressão dos genes Oct-4 e STAT-3 por RT-PCR. As NPM revestidas com ácido láurico, citrato e DMSA não apresentaram citotoxicidade, a julgar pelos altos valores de IC50 encontrados (254, 433 e 2260 μg de ferro/mL, respectivamente), sendo que as nanoparticulas revestidas com citrato foram as escolhidas para serem utilizadas como suporte para expandir as células mES. O tempo de duplicação para as células cultivadas na presença da NPM foi ligeiramente maior do que na presença com co-cultura de MEF (20,67 ± 0,19 vs. 15,95 ± 0,21) (p= 0,001). A morfologia das células mES cultivadas na presença da NPM foi semelhante à de células ES, sendo que, a sua pluripotência foi confirmada pela expressão dos marcadores de indiferenciação Oct-4 e Nanog por RT-PCR e da alta atividade de fosfatase alcalina. Uma linhagem de células tronco embrionárias bovinas (bES) foi obtida e mantida por seis subcultivos por período de 60 dias. A pluripotência das células bES foi confirmada morfologicamente, bem como pela expressão dos genes do estado indiferenciado Oct-4 e STAT-3Made available in DSpace on 2014-07-29T15:10:32Z (GMT). No. of bitstreams: 1 Tese-Erika2009.pdf: 3993118 bytes, checksum: 2852e36019237acb54dcbae5e0aa2faf (MD5) Previous issue date: 2009-08-14application/pdfhttp://repositorio.bc.ufg.br/TEDE/retrieve/3611/Tese-Erika2009.pdf.jpgporUniversidade Federal de GoiásDoutorado em BiologiaUFGBRCiencias BiologicasCélulas tronco embrionáriasNanopartículas magnéticasPluripotênciaEmbryonic stem cellsMagnetic nanoparticlesPluripotencyCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULARCaracterização do estado indiferenciado de células tronco embrionárias murinas expandidas na presença de nanopartículas magnéticas e isolamento de células tronco embrionárias a partir de blastócitos bovinosCharacterization of undifferentiated state of murine embryonic stem cells expanded in the presence of magnetic nanoparticles and isolation of embryonic stem cells from bovine blastocystsinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFGinstname:Universidade Federal de Goiás (UFG)instacron:UFGORIGINALTese-Erika2009.pdfapplication/pdf3993118http://repositorio.bc.ufg.br/tede/bitstreams/2a6bb244-60bc-4ddc-9bc2-cd6b6d98fbf5/download2852e36019237acb54dcbae5e0aa2fafMD51THUMBNAILTese-Erika2009.pdf.jpgTese-Erika2009.pdf.jpgGenerated Thumbnailimage/jpeg3637http://repositorio.bc.ufg.br/tede/bitstreams/9076e0a2-b4bb-4c18-b05a-84f6e5ded593/download84a51af7728c60c13a4aa906111ca431MD52tde/10142014-07-30 03:06:56.058open.accessoai:repositorio.bc.ufg.br:tde/1014http://repositorio.bc.ufg.br/tedeRepositório InstitucionalPUBhttp://repositorio.bc.ufg.br/oai/requesttasesdissertacoes.bc@ufg.bropendoar:2014-07-30T06:06:56Repositório Institucional da UFG - Universidade Federal de Goiás (UFG)false |
dc.title.por.fl_str_mv |
Caracterização do estado indiferenciado de células tronco embrionárias murinas expandidas na presença de nanopartículas magnéticas e isolamento de células tronco embrionárias a partir de blastócitos bovinos |
dc.title.alternative.eng.fl_str_mv |
Characterization of undifferentiated state of murine embryonic stem cells expanded in the presence of magnetic nanoparticles and isolation of embryonic stem cells from bovine blastocysts |
title |
Caracterização do estado indiferenciado de células tronco embrionárias murinas expandidas na presença de nanopartículas magnéticas e isolamento de células tronco embrionárias a partir de blastócitos bovinos |
spellingShingle |
Caracterização do estado indiferenciado de células tronco embrionárias murinas expandidas na presença de nanopartículas magnéticas e isolamento de células tronco embrionárias a partir de blastócitos bovinos FREITAS, Erika Regina Leal de Células tronco embrionárias Nanopartículas magnéticas Pluripotência Embryonic stem cells Magnetic nanoparticles Pluripotency CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
title_short |
Caracterização do estado indiferenciado de células tronco embrionárias murinas expandidas na presença de nanopartículas magnéticas e isolamento de células tronco embrionárias a partir de blastócitos bovinos |
title_full |
Caracterização do estado indiferenciado de células tronco embrionárias murinas expandidas na presença de nanopartículas magnéticas e isolamento de células tronco embrionárias a partir de blastócitos bovinos |
title_fullStr |
Caracterização do estado indiferenciado de células tronco embrionárias murinas expandidas na presença de nanopartículas magnéticas e isolamento de células tronco embrionárias a partir de blastócitos bovinos |
title_full_unstemmed |
Caracterização do estado indiferenciado de células tronco embrionárias murinas expandidas na presença de nanopartículas magnéticas e isolamento de células tronco embrionárias a partir de blastócitos bovinos |
title_sort |
Caracterização do estado indiferenciado de células tronco embrionárias murinas expandidas na presença de nanopartículas magnéticas e isolamento de células tronco embrionárias a partir de blastócitos bovinos |
author |
FREITAS, Erika Regina Leal de |
author_facet |
FREITAS, Erika Regina Leal de |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
GUILLO, Lídia Andreu |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/3401436781775091 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/5011536994328123 |
dc.contributor.author.fl_str_mv |
FREITAS, Erika Regina Leal de |
contributor_str_mv |
GUILLO, Lídia Andreu |
dc.subject.por.fl_str_mv |
Células tronco embrionárias Nanopartículas magnéticas Pluripotência |
topic |
Células tronco embrionárias Nanopartículas magnéticas Pluripotência Embryonic stem cells Magnetic nanoparticles Pluripotency CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
dc.subject.eng.fl_str_mv |
Embryonic stem cells Magnetic nanoparticles Pluripotency |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
description |
Magnetic nanoparticles (MNPs) have been used in a great variety of biomedical applications, especially in cancer treatment, drug delivery, and diagnosis by magnetic resonance imaging. Embryonic stem cells (ESCs), due to its capacity of auto-renewal and differentiation in some types of cells, offer a great potential for its use in tissue regeneration and in alternative treatments for many degenerative diseases. The study had as aims: i) to evaluate in vitro citotoxicity of maghemite nanoparticles functionalized with lauric acid, DMSA, and citrate in human melanoma cells (SK-MEL-37) by the MTT assay, electronic microscopy, and DNA electroforesis by agarose gel; ii) to develop a culture system using the previously selected MNPs and a magnet to expand in vitro murine embryonic stem cells (mESCs) in the absence of a co-culture of murine embryonic fibroblasts (MEF) (the indifferentiated state of the mES was analyzed by alkaline phosphatase cytochemistry, electronic microscopy, and analysis of Oct-4 and Nanog gene expression by RT-PCR); iii) to isolate and expand ESCs from bovine blastocysts, and to characterize its pluripotency by analysis of Oct-4 and STAT-3 gene expression by RT-PCR. The MNPs coated with lauric acid, citrate, and DMSA showed no citotoxicity, judging by the high values of IC50 found (254, 433 and 2260 μg-iron/ml, respectively), and that the nanoparticles coated with citrate was chosen to expand the mESCs. The doubling time for the cells cultivated in the presence of MNPs was slightly higher than in the presence of MEF (20.67 ± 0.19 vs. 15.95 ± 0.21) (p= 0.001). The mESCs cultivated in the presence of MNPs showed morphology similar to ESCs, and its pluripotency was confirmed by expression of indifferentiation markers Oct-4 and Nanog by RT-PCR and high alkaline phosphatase activity. One bovine embryonic stem cell (bESCs) line was obtained and maintained by six subcultures for 60 days period. The pluripotency of the bESCs was confirmed morphologically as well as by Oct-4 e STAT-3 gene expression |
publishDate |
2009 |
dc.date.issued.fl_str_mv |
2009-08-14 |
dc.date.available.fl_str_mv |
2010-02-05 |
dc.date.accessioned.fl_str_mv |
2014-07-29T15:10:32Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
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publishedVersion |
dc.identifier.citation.fl_str_mv |
FREITAS, Erika Regina Leal de. Characterization of undifferentiated state of murine embryonic stem cells expanded in the presence of magnetic nanoparticles and isolation of embryonic stem cells from bovine blastocysts. 2009. 127 f. Tese (Doutorado em Ciencias Biologicas) - Universidade Federal de Goiás, Goiânia, 2009. |
dc.identifier.uri.fl_str_mv |
http://repositorio.bc.ufg.br/tede/handle/tde/1014 |
identifier_str_mv |
FREITAS, Erika Regina Leal de. Characterization of undifferentiated state of murine embryonic stem cells expanded in the presence of magnetic nanoparticles and isolation of embryonic stem cells from bovine blastocysts. 2009. 127 f. Tese (Doutorado em Ciencias Biologicas) - Universidade Federal de Goiás, Goiânia, 2009. |
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http://repositorio.bc.ufg.br/tede/handle/tde/1014 |
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Universidade Federal de Goiás |
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Doutorado em Biologia |
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UFG |
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BR |
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Ciencias Biologicas |
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Universidade Federal de Goiás |
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