Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC

Detalhes bibliográficos
Autor(a) principal: Silva, Gleyce Oliveira [UNESP]
Data de Publicação: 2015
Tipo de documento: Tese
Idioma: por
Título da fonte: Repositório Institucional da UNESP
Texto Completo: http://hdl.handle.net/11449/136702
http://www.athena.biblioteca.unesp.br/exlibris/bd/cathedra/28-03-2016/000860768.pdf
Resumo: The aim of this study was to evaluate the function of Wnt/β-catenin signaling through LRP-6 and Frizzled-6 receptors in the differentiation of dental pulp stem cells from permanent teeth (DPSC) into endothelial cells. DPSC were transduced with EGFP-tagged lentiviral shRNA vectors (LRP6, Frizzled6 or empty vector - control) for experiments. In vitro assay evaluated GSK3-β and β-catenin expression by western blot in rhWnt1 and rhVEGF165 presence, and VEGF and CXCL-8 (IL-8) expression by ELISA. Endothelial markers expression (western blot and PCR) and tube formation were analyzed after endothelial differentiation of DPSCs. In vivo, tooth slices/scaffolds seeded with transduced DPSCs were implanted subcutaneously in back of immunodefficient mice and blood vessels were counted per immunohistochemistry for eGFP and HE staining. Active β- catenin was more expressed in shRNA-LRP6 and shRNA-Frizzled6 than in control cells, and increased with rhVEGF165 and rhWnt1 supplementation. Phosphorylated GSK3-β expression was lower, however also increased or maintained with rhVEGF165 or rhWnt1. VEGF expression was higher in shRNA-Frizzled6 than in control and shRNA-LRP6 (p<0,05), IL8 expression was lower in shRNA-LRP6, with statistically difference of the others cells. Endothelial markers CD31 and VEGFR2 expression decreased in shRNA-LRP6, but VEGFR2 expression increased in shRNAFrizzled6. shRNA-Frizzled6 and shRNA-LRP6 tube formation was lower when compared to control, however shRNA-Frizzled6 had tendency to increase proliferation in 144h. In vivo, shRNA-LRP6 showed fewer blood vessels formed than other cells (p<0,05). Collectively, the results of this study suggest that Wnt/β-catenin signaling regulates endothelial differentiation of DPSC through LRP6
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spelling Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSCEngenharia tecidualVasos sanguineosCélulas-troncoTissue engineeringThe aim of this study was to evaluate the function of Wnt/β-catenin signaling through LRP-6 and Frizzled-6 receptors in the differentiation of dental pulp stem cells from permanent teeth (DPSC) into endothelial cells. DPSC were transduced with EGFP-tagged lentiviral shRNA vectors (LRP6, Frizzled6 or empty vector - control) for experiments. In vitro assay evaluated GSK3-β and β-catenin expression by western blot in rhWnt1 and rhVEGF165 presence, and VEGF and CXCL-8 (IL-8) expression by ELISA. Endothelial markers expression (western blot and PCR) and tube formation were analyzed after endothelial differentiation of DPSCs. In vivo, tooth slices/scaffolds seeded with transduced DPSCs were implanted subcutaneously in back of immunodefficient mice and blood vessels were counted per immunohistochemistry for eGFP and HE staining. Active β- catenin was more expressed in shRNA-LRP6 and shRNA-Frizzled6 than in control cells, and increased with rhVEGF165 and rhWnt1 supplementation. Phosphorylated GSK3-β expression was lower, however also increased or maintained with rhVEGF165 or rhWnt1. VEGF expression was higher in shRNA-Frizzled6 than in control and shRNA-LRP6 (p<0,05), IL8 expression was lower in shRNA-LRP6, with statistically difference of the others cells. Endothelial markers CD31 and VEGFR2 expression decreased in shRNA-LRP6, but VEGFR2 expression increased in shRNAFrizzled6. shRNA-Frizzled6 and shRNA-LRP6 tube formation was lower when compared to control, however shRNA-Frizzled6 had tendency to increase proliferation in 144h. In vivo, shRNA-LRP6 showed fewer blood vessels formed than other cells (p<0,05). Collectively, the results of this study suggest that Wnt/β-catenin signaling regulates endothelial differentiation of DPSC through LRP6O objetivo deste estudo foi avaliar a função da via de sinalização Wnt/β- catenina através dos receptores LRP6 e Frizzled6 na diferenciação de células-tronco de polpa dental de dentes permanentes (DPSC) em células endoteliais. DPSC foram transduzidas com marcadores eGFP e vetores lentivirais shRNA (LRP6, Frizzled6 ou vetor vazio - controle) para os experimentos. Os testes in vitro avaliaram a expressão de GSK3-β e β- catenina por western blot na presença de rhWnt1 e rhVEGF165 e de VEGF e CXCL-8 (IL-8) por ELISA. A expressão de marcadores endoteliais (western blot e PCR) e formação de túbulos capilares foram analisados após a diferenciação endotelial das DPSCs. In vivo, fatias dentárias/matrizes condutivas semeadas com DPSCs-shRNA foram implantadas em subcutâneo de dorso de camundongos imunodeprimidos por 28 dias e o número de vasos sanguíneos foi determinado por imunohistoquímica para eGFP e coloração por HE. β-catenina ativa foi mais expressa em shRNA-LRP6 e shRNA-Frizzled6 que nas células controle, e sua expressão aumentou com a suplementação com rhVEGF165 e rhWnt1. A expressão de GSK3-β fosforilado foi menor, porém também aumenta ou permanece estável com rhVEGF165 ou rhWnt1. Quanto à expressão de VEGF, em shRNA-Frizzled6 foi maior que nas células controle e em shRNA-LRP6 (p<0,05), enquanto que a expressão de IL8 foi menor em shRNA-LRP6, diferindo estatisticamente das outras células. A expressão dos marcadores endoteliais CD31 e VEGFR2 diminuiu nas células shRNA-LRP6, enquanto que em shRNAFrizzled6 a expressão de VEGFR2 foi aumentada. A formação de túbulos capilares de shRNA-Frizzled6 e shRNA-LRP6 foi menor quando comparado ao controle, porém shRNA-Frizzled6 obteve uma tendência de aumento na proliferação de capilares em 144h. In vivo, DPSC-shRNALRP6 apresentou menor número de capilares formados quando comparados com as outras duas...Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Estadual Paulista (Unesp)Camargo, Carlos Henrique Ribeiro [UNESP]Universidade Estadual Paulista (Unesp)Silva, Gleyce Oliveira [UNESP]2016-04-01T17:54:51Z2016-04-01T17:54:51Z2015-07-31info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis91 f. : il.application/pdfSILVA, Gleyce Oliveira. Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC. 2015. 91 f. Tese (doutorado) - UNESP - Univ Estadual Paulista, Instituto de Ciência e Tecnologia de São José dos Campos, 2015.http://hdl.handle.net/11449/136702000860768http://www.athena.biblioteca.unesp.br/exlibris/bd/cathedra/28-03-2016/000860768.pdf33004145070P8Alephreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPporinfo:eu-repo/semantics/openAccess2023-10-12T06:06:19Zoai:repositorio.unesp.br:11449/136702Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462023-10-12T06:06:19Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false
dc.title.none.fl_str_mv Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC
title Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC
spellingShingle Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC
Silva, Gleyce Oliveira [UNESP]
Engenharia tecidual
Vasos sanguineos
Células-tronco
Tissue engineering
title_short Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC
title_full Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC
title_fullStr Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC
title_full_unstemmed Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC
title_sort Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC
author Silva, Gleyce Oliveira [UNESP]
author_facet Silva, Gleyce Oliveira [UNESP]
author_role author
dc.contributor.none.fl_str_mv Camargo, Carlos Henrique Ribeiro [UNESP]
Universidade Estadual Paulista (Unesp)
dc.contributor.author.fl_str_mv Silva, Gleyce Oliveira [UNESP]
dc.subject.por.fl_str_mv Engenharia tecidual
Vasos sanguineos
Células-tronco
Tissue engineering
topic Engenharia tecidual
Vasos sanguineos
Células-tronco
Tissue engineering
description The aim of this study was to evaluate the function of Wnt/β-catenin signaling through LRP-6 and Frizzled-6 receptors in the differentiation of dental pulp stem cells from permanent teeth (DPSC) into endothelial cells. DPSC were transduced with EGFP-tagged lentiviral shRNA vectors (LRP6, Frizzled6 or empty vector - control) for experiments. In vitro assay evaluated GSK3-β and β-catenin expression by western blot in rhWnt1 and rhVEGF165 presence, and VEGF and CXCL-8 (IL-8) expression by ELISA. Endothelial markers expression (western blot and PCR) and tube formation were analyzed after endothelial differentiation of DPSCs. In vivo, tooth slices/scaffolds seeded with transduced DPSCs were implanted subcutaneously in back of immunodefficient mice and blood vessels were counted per immunohistochemistry for eGFP and HE staining. Active β- catenin was more expressed in shRNA-LRP6 and shRNA-Frizzled6 than in control cells, and increased with rhVEGF165 and rhWnt1 supplementation. Phosphorylated GSK3-β expression was lower, however also increased or maintained with rhVEGF165 or rhWnt1. VEGF expression was higher in shRNA-Frizzled6 than in control and shRNA-LRP6 (p<0,05), IL8 expression was lower in shRNA-LRP6, with statistically difference of the others cells. Endothelial markers CD31 and VEGFR2 expression decreased in shRNA-LRP6, but VEGFR2 expression increased in shRNAFrizzled6. shRNA-Frizzled6 and shRNA-LRP6 tube formation was lower when compared to control, however shRNA-Frizzled6 had tendency to increase proliferation in 144h. In vivo, shRNA-LRP6 showed fewer blood vessels formed than other cells (p<0,05). Collectively, the results of this study suggest that Wnt/β-catenin signaling regulates endothelial differentiation of DPSC through LRP6
publishDate 2015
dc.date.none.fl_str_mv 2015-07-31
2016-04-01T17:54:51Z
2016-04-01T17:54:51Z
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 SILVA, Gleyce Oliveira. Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC. 2015. 91 f. Tese (doutorado) - UNESP - Univ Estadual Paulista, Instituto de Ciência e Tecnologia de São José dos Campos, 2015.
http://hdl.handle.net/11449/136702
000860768
http://www.athena.biblioteca.unesp.br/exlibris/bd/cathedra/28-03-2016/000860768.pdf
33004145070P8
identifier_str_mv SILVA, Gleyce Oliveira. Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC. 2015. 91 f. Tese (doutorado) - UNESP - Univ Estadual Paulista, Instituto de Ciência e Tecnologia de São José dos Campos, 2015.
000860768
33004145070P8
url http://hdl.handle.net/11449/136702
http://www.athena.biblioteca.unesp.br/exlibris/bd/cathedra/28-03-2016/000860768.pdf
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 91 f. : il.
application/pdf
dc.publisher.none.fl_str_mv Universidade Estadual Paulista (Unesp)
publisher.none.fl_str_mv Universidade Estadual Paulista (Unesp)
dc.source.none.fl_str_mv Aleph
reponame:Repositório Institucional da UNESP
instname:Universidade Estadual Paulista (UNESP)
instacron:UNESP
instname_str Universidade Estadual Paulista (UNESP)
instacron_str UNESP
institution UNESP
reponame_str Repositório Institucional da UNESP
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repository.name.fl_str_mv Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)
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