Efeitos do LRP6 e Frizzled6 na diferenciação endotelial de DPSC
Autor(a) principal: | |
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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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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 |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
_version_ |
1799964532443971584 |