Efeito da suplementa??o de leucina sobre a prolifera??o de pr?-osteoblastos da linhagem MC3T3-E1
Ano de defesa: | 2016 |
---|---|
Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | |
Tipo de documento: | Tese |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Pontif?cia Universidade Cat?lica do Rio Grande do Sul
|
Programa de Pós-Graduação: |
Programa de P?s-Gradua??o em Medicina e Ci?ncias da Sa?de
|
Departamento: |
Faculdade de Medicina
|
País: |
Brasil
|
Palavras-chave em Português: | |
Área do conhecimento CNPq: | |
Link de acesso: | http://tede2.pucrs.br/tede2/handle/tede/6852 |
Resumo: | Introduction: leucine (Leu) is an essential branched-chain amino acid, present in dairy products, which has been investigated for exert an important role in cell signaling. However, most studies evaluating cellular responses mediated by Leu, works within a normal perspective in AA supply and little is known about the effects that supplementation can generate on the cell proliferation mechanisms. The effects of excess of this amino acid, have been extensively studied in many cell types, but there is an important limitation on the amount of information available in the scientific literature regarding their actions in bone cells. Objective: the aim of this study to determine the effects of leucine supplementation on proliferation of pre-osteoblasts of the MC3T3-E1 lineage. Methods: the MC3T3-E1 cells were kept in ?-MEM supplemented with 10% fetal bovine serum and 1% antibiotic. After initial determination of concentrations, the cells were treated during 48 hours, by the addition of 50 ?M Leu, which corresponds to 12,5% in addition of the amino acid to the culture medium. Untreated cells represented the control group. The evaluation of the viability and proliferation of cultured cells was performed with Trypan Blue dye (0.4%). To identify the mechanisms related to decreased cellular proliferation, assays were performed to verify cytotoxicity (LDH); apotosis (Annexin V); oxidative stress (TBARS and DCFH); inflammation (TGF-? 1 and CBA); autophagy (acridine orange and flow cytometry); senescence (DAPI and flow cytometry); and DNA damage (alkaline comet assay). Results and conclusions: Leu supplementation (50 ?M) decreases cell proliferation by 40% with causes not related to cell necrosis, apoptosis, oxidative stress, inflammation or autophagy. The Leu supplementation caused DNA damage, with consequent increase in senescence and decrease of proliferation of MC3T3-E1 cells. |
id |
P_RS_856d845a084ce5cb0763b7d3906278bb |
---|---|
oai_identifier_str |
oai:tede2.pucrs.br:tede/6852 |
network_acronym_str |
P_RS |
network_name_str |
Biblioteca Digital de Teses e Dissertações da PUC_RS |
repository_id_str |
|
spelling |
Oliveira, Jarbas Rodrigues de140.906.430-15http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781448U1886.487.020-20http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4737483T8Luz, Raquel Dias da2016-07-26T14:11:19Z2016-03-31http://tede2.pucrs.br/tede2/handle/tede/6852Introduction: leucine (Leu) is an essential branched-chain amino acid, present in dairy products, which has been investigated for exert an important role in cell signaling. However, most studies evaluating cellular responses mediated by Leu, works within a normal perspective in AA supply and little is known about the effects that supplementation can generate on the cell proliferation mechanisms. The effects of excess of this amino acid, have been extensively studied in many cell types, but there is an important limitation on the amount of information available in the scientific literature regarding their actions in bone cells. Objective: the aim of this study to determine the effects of leucine supplementation on proliferation of pre-osteoblasts of the MC3T3-E1 lineage. Methods: the MC3T3-E1 cells were kept in ?-MEM supplemented with 10% fetal bovine serum and 1% antibiotic. After initial determination of concentrations, the cells were treated during 48 hours, by the addition of 50 ?M Leu, which corresponds to 12,5% in addition of the amino acid to the culture medium. Untreated cells represented the control group. The evaluation of the viability and proliferation of cultured cells was performed with Trypan Blue dye (0.4%). To identify the mechanisms related to decreased cellular proliferation, assays were performed to verify cytotoxicity (LDH); apotosis (Annexin V); oxidative stress (TBARS and DCFH); inflammation (TGF-? 1 and CBA); autophagy (acridine orange and flow cytometry); senescence (DAPI and flow cytometry); and DNA damage (alkaline comet assay). Results and conclusions: Leu supplementation (50 ?M) decreases cell proliferation by 40% with causes not related to cell necrosis, apoptosis, oxidative stress, inflammation or autophagy. The Leu supplementation caused DNA damage, with consequent increase in senescence and decrease of proliferation of MC3T3-E1 cells.Introdu??o: a leucina (Leu) ? um amino?cido (AA) essencial, de cadeia ramificada, presente na alimenta??o humana, principalmente no leite e seus derivados, que tem sido investigado por exercer um importante papel na sinaliza??o celular. Contudo, a maioria dos estudos que avalia as respostas celulares mediadas pela Leu, trabalha dentro de uma perspectiva de normalidade na oferta do AA e pouco se sabe sobre os efeitos que a suplementa??o pode gerar sobre os mecanismos de prolifera??o celular. Os efeitos do excesso deste amino?cido, t?m sido extensivamente estudado em diversos tipos de c?lulas, entretanto existe uma limita??o importante na quantidade de informa??es dispon?veis na literatura cient?fica em rela??o as suas a??es em c?lulas do tecido ?sseo. Objetivo: este estudo teve como objetivo analisar os efeitos da suplementa??o de leucina sobre a prolifera??o de pr?-osteoblastos da linhagem MC3T3-E1. M?todos: a cultura dos pr?-osteoblastos da linhagem MC3T3-E1, foi realizada com ?-MEM, suplementado com 10% de soro fetal bovino e 1% de antibi?tico. Ap?s determina??o de concentra??es, o tratamento foi feito com a adi??o de Leu, dilu?da ao meio de cultura nas concentra??es de 50 ?M, o que corresponde a um acr?scimo percentual de 12.5% a mais do amino?cido ao meio de cultura, por 48 horas. A viabilidade e a prolifera??o celular foram avaliadas pela t?cnica do Trypan Blue. Para a identifica??o dos mecanismos relacionados a inibi??o da prolifera??o celular, foram realizados ensaios que avaliaram a citotoxicidade (LDH); apoptose (Anexina V); estresse oxidativo (TBARS e DCFH); perfil inflamat?rio (TGF-? 1 e CBA); autofagia (laranja de acridina e citometria de fluxo); senesc?ncia (DAPI) e dano ao DNA (teste cometa). Resultados e conclus?es: a suplementa??o de Leu (50 ?M) inibe a prolifera??o celular em 40%, com causas n?o relacionadas a necrose celular, apoptose, estresse oxidativo, inflama??o ou autofagia. A suplementa??o de Leu provocou dano ao DNA, com consequente senesc?ncia e diminui??o da prolifera??o celular de pr?-osteoblastos da linhagem MC3T3-E1.Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-07-26T14:11:19Z No. of bitstreams: 1 TES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf: 2184806 bytes, checksum: 196521807ad1dcdfa8bffc204202abe6 (MD5)Made available in DSpace on 2016-07-26T14:11:19Z (GMT). No. of bitstreams: 1 TES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf: 2184806 bytes, checksum: 196521807ad1dcdfa8bffc204202abe6 (MD5) Previous issue date: 2016-03-31Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPESapplication/pdfhttp://tede2.pucrs.br:80/tede2/retrieve/165819/TES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf.jpgporPontif?cia Universidade Cat?lica do Rio Grande do SulPrograma de P?s-Gradua??o em Medicina e Ci?ncias da Sa?dePUCRSBrasilFaculdade de MedicinaDIETOTERAPIAPR?-OSTEOBLASTOSTECIDO ?SSEOMETABOLISMONUTRI??OCIENCIAS DA SAUDE::MEDICINAEfeito da suplementa??o de leucina sobre a prolifera??o de pr?-osteoblastos da linhagem MC3T3-E1Effect of leucine supplementation on pre-osteoblasts MC3T3-E1 cell lineage proliferationinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis7620745074616285884600600600600-8624664729441623247-9693694523087866272075167498588264571info:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da PUC_RSinstname:Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS)instacron:PUC_RSTHUMBNAILTES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf.jpgTES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf.jpgimage/jpeg2850http://tede2.pucrs.br/tede2/bitstream/tede/6852/5/TES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf.jpg68b6815ffbfb0dfe1280ef1042207d41MD55TEXTTES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf.txtTES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf.txttext/plain130826http://tede2.pucrs.br/tede2/bitstream/tede/6852/4/TES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf.txteac500fe0a6bdd0a5dbe173fd7fcd3d0MD54LICENSElicense.txtlicense.txttext/plain; charset=utf-8610http://tede2.pucrs.br/tede2/bitstream/tede/6852/3/license.txt5a9d6006225b368ef605ba16b4f6d1beMD53ORIGINALTES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdfTES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdfapplication/pdf2184806http://tede2.pucrs.br/tede2/bitstream/tede/6852/2/TES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf196521807ad1dcdfa8bffc204202abe6MD52tede/68522016-07-26 20:00:28.089oai:tede2.pucrs.br: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Biblioteca Digital de Teses e Dissertaçõeshttp://tede2.pucrs.br/tede2/PRIhttps://tede2.pucrs.br/oai/requestbiblioteca.central@pucrs.br||opendoar:2016-07-26T23:00:28Biblioteca Digital de Teses e Dissertações da PUC_RS - Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS)false |
dc.title.por.fl_str_mv |
Efeito da suplementa??o de leucina sobre a prolifera??o de pr?-osteoblastos da linhagem MC3T3-E1 |
dc.title.alternative.eng.fl_str_mv |
Effect of leucine supplementation on pre-osteoblasts MC3T3-E1 cell lineage proliferation |
title |
Efeito da suplementa??o de leucina sobre a prolifera??o de pr?-osteoblastos da linhagem MC3T3-E1 |
spellingShingle |
Efeito da suplementa??o de leucina sobre a prolifera??o de pr?-osteoblastos da linhagem MC3T3-E1 Luz, Raquel Dias da DIETOTERAPIA PR?-OSTEOBLASTOS TECIDO ?SSEO METABOLISMO NUTRI??O CIENCIAS DA SAUDE::MEDICINA |
title_short |
Efeito da suplementa??o de leucina sobre a prolifera??o de pr?-osteoblastos da linhagem MC3T3-E1 |
title_full |
Efeito da suplementa??o de leucina sobre a prolifera??o de pr?-osteoblastos da linhagem MC3T3-E1 |
title_fullStr |
Efeito da suplementa??o de leucina sobre a prolifera??o de pr?-osteoblastos da linhagem MC3T3-E1 |
title_full_unstemmed |
Efeito da suplementa??o de leucina sobre a prolifera??o de pr?-osteoblastos da linhagem MC3T3-E1 |
title_sort |
Efeito da suplementa??o de leucina sobre a prolifera??o de pr?-osteoblastos da linhagem MC3T3-E1 |
author |
Luz, Raquel Dias da |
author_facet |
Luz, Raquel Dias da |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Oliveira, Jarbas Rodrigues de |
dc.contributor.advisor1ID.fl_str_mv |
140.906.430-15 |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781448U1 |
dc.contributor.authorID.fl_str_mv |
886.487.020-20 |
dc.contributor.authorLattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4737483T8 |
dc.contributor.author.fl_str_mv |
Luz, Raquel Dias da |
contributor_str_mv |
Oliveira, Jarbas Rodrigues de |
dc.subject.por.fl_str_mv |
DIETOTERAPIA PR?-OSTEOBLASTOS TECIDO ?SSEO METABOLISMO NUTRI??O |
topic |
DIETOTERAPIA PR?-OSTEOBLASTOS TECIDO ?SSEO METABOLISMO NUTRI??O CIENCIAS DA SAUDE::MEDICINA |
dc.subject.cnpq.fl_str_mv |
CIENCIAS DA SAUDE::MEDICINA |
description |
Introduction: leucine (Leu) is an essential branched-chain amino acid, present in dairy products, which has been investigated for exert an important role in cell signaling. However, most studies evaluating cellular responses mediated by Leu, works within a normal perspective in AA supply and little is known about the effects that supplementation can generate on the cell proliferation mechanisms. The effects of excess of this amino acid, have been extensively studied in many cell types, but there is an important limitation on the amount of information available in the scientific literature regarding their actions in bone cells. Objective: the aim of this study to determine the effects of leucine supplementation on proliferation of pre-osteoblasts of the MC3T3-E1 lineage. Methods: the MC3T3-E1 cells were kept in ?-MEM supplemented with 10% fetal bovine serum and 1% antibiotic. After initial determination of concentrations, the cells were treated during 48 hours, by the addition of 50 ?M Leu, which corresponds to 12,5% in addition of the amino acid to the culture medium. Untreated cells represented the control group. The evaluation of the viability and proliferation of cultured cells was performed with Trypan Blue dye (0.4%). To identify the mechanisms related to decreased cellular proliferation, assays were performed to verify cytotoxicity (LDH); apotosis (Annexin V); oxidative stress (TBARS and DCFH); inflammation (TGF-? 1 and CBA); autophagy (acridine orange and flow cytometry); senescence (DAPI and flow cytometry); and DNA damage (alkaline comet assay). Results and conclusions: Leu supplementation (50 ?M) decreases cell proliferation by 40% with causes not related to cell necrosis, apoptosis, oxidative stress, inflammation or autophagy. The Leu supplementation caused DNA damage, with consequent increase in senescence and decrease of proliferation of MC3T3-E1 cells. |
publishDate |
2016 |
dc.date.accessioned.fl_str_mv |
2016-07-26T14:11:19Z |
dc.date.issued.fl_str_mv |
2016-03-31 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://tede2.pucrs.br/tede2/handle/tede/6852 |
url |
http://tede2.pucrs.br/tede2/handle/tede/6852 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.program.fl_str_mv |
7620745074616285884 |
dc.relation.confidence.fl_str_mv |
600 600 600 600 |
dc.relation.department.fl_str_mv |
-8624664729441623247 |
dc.relation.cnpq.fl_str_mv |
-969369452308786627 |
dc.relation.sponsorship.fl_str_mv |
2075167498588264571 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Pontif?cia Universidade Cat?lica do Rio Grande do Sul |
dc.publisher.program.fl_str_mv |
Programa de P?s-Gradua??o em Medicina e Ci?ncias da Sa?de |
dc.publisher.initials.fl_str_mv |
PUCRS |
dc.publisher.country.fl_str_mv |
Brasil |
dc.publisher.department.fl_str_mv |
Faculdade de Medicina |
publisher.none.fl_str_mv |
Pontif?cia Universidade Cat?lica do Rio Grande do Sul |
dc.source.none.fl_str_mv |
reponame:Biblioteca Digital de Teses e Dissertações da PUC_RS instname:Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) instacron:PUC_RS |
instname_str |
Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) |
instacron_str |
PUC_RS |
institution |
PUC_RS |
reponame_str |
Biblioteca Digital de Teses e Dissertações da PUC_RS |
collection |
Biblioteca Digital de Teses e Dissertações da PUC_RS |
bitstream.url.fl_str_mv |
http://tede2.pucrs.br/tede2/bitstream/tede/6852/5/TES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf.jpg http://tede2.pucrs.br/tede2/bitstream/tede/6852/4/TES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf.txt http://tede2.pucrs.br/tede2/bitstream/tede/6852/3/license.txt http://tede2.pucrs.br/tede2/bitstream/tede/6852/2/TES_RAQUEL_DA_LUZ_DIAS_COMPLETO.pdf |
bitstream.checksum.fl_str_mv |
68b6815ffbfb0dfe1280ef1042207d41 eac500fe0a6bdd0a5dbe173fd7fcd3d0 5a9d6006225b368ef605ba16b4f6d1be 196521807ad1dcdfa8bffc204202abe6 |
bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 MD5 |
repository.name.fl_str_mv |
Biblioteca Digital de Teses e Dissertações da PUC_RS - Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) |
repository.mail.fl_str_mv |
biblioteca.central@pucrs.br|| |
_version_ |
1796793221514264576 |