Avaliação molecular e imunohistoquímica da uretra de ratas após trauma induzido e terapia intravenosa com células-tronco derivadas de músculo

Detalhes bibliográficos
Autor(a) principal: Bilhar, Andreisa Paiva Monteiro [UNIFESP]
Data de Publicação: 2017
Tipo de documento: Tese
Idioma: por
Título da fonte: Repositório Institucional da UNIFESP
Texto Completo: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=5760899
http://repositorio.unifesp.br/handle/11600/49880
Resumo: Objective: To identify the migration to the urethra of muscle derived stem cells (MDSCs) after intravenous (IV) injection in rats submitted to pelvic floor trauma due to vaginal distension (DV) and to analyze the effects of MDSC in the urethra of rats after seven and thirty days of the trauma in regards to: (1) mRNA expression of collagens, vascular endothelial growth factor (VEGF), nerve growth factor (NGF), Ki67 cell proliferation marker, and the expression of genes related to smooth and striated muscle apparatus; (2) expression of smooth and striated muscle proteins. Methods: MDSCs expressing green fluorescent protein (GFP) were injected into the tail vein of the rats at day three after DV. The urethras of five groups were analyzed: Control, Trauma 7 days (animals submitted to DV, sacrificed 7 days), MDSC 7 days (animals submitted to DV receiving IV therapy with MDSC, sacrificed 7 days), Trauma 30 days (animals submitted to DV, sacrificed at 30 days), MDSC 30 days (animals submitted to DV receiving IV therapy with MDSC, sacrificed at 30 days). Location of GFP cells was verified at 2 hours and 7 days after IV injection. RT-qPCR was used for gene expression and immunohistochemistry for protein identification and quantification. Results: MDSCs were early identified in the urethra, but only deformed cells after 7 days. After seven days of trauma, the MYH11 gene was overexpressed in the Trauma group in relation to Control (p=0.01). Expression of the mKi67 gene was increased in the MDSC group relative to Trauma (p=0.03) and Control (p=0.02). Expression of the COL1A1 and COL3A1 genes increased in the MDSC group relative to the Control (p=0.001 and p=0.002). The gene expression of NGF was decreased in the MDSC group in relation to Trauma (p=0.001) and there was no difference in expression of the MYH2, Desmin and VEGF gene. In the same period, immunohistochemical expression of MHY11, MYH2 and Desmin was increased in the MDSC group in relation to Trauma (p=0.01, p=0.005 and p=0.02) and decreased in the Trauma group in relation to the Control (p =0.01, p=0.008 and p=0.03). After 30 days of trauma, MYH11 and MYH2 gene expression was increased in the Trauma group compared to Control (p=0.03 and p=0.04). Expression of the mKi67 gene was increased in the MDSC group relative to Trauma (p=0.02) and Control (p=0.01). Expression of the COL1A1 gene increased in the MDSC group compared to the Control (p=0.008) and Trauma (p=0.03) and COL3A1 gene expression increased in the MDSC group compared to Control (p=0.03). However, the gene expression of NGF was decreased in the MDSC group in relation to Trauma (p=0.002) and there was no difference in the gene expression of Desmin and VEGF. In the same period, immunohistochemical expression of MYH11, MYH2 and Desmin was increased in the MDSC group in relation to Trauma (p=0.01, p=0.002 and p=0.01) and MYH2 gene expression decreased in the Trauma group compared to Control (p=0.04). Conclusion: MDSCs migrated early to the traumatized urethra, but did not integrate into the tissue. IV administration of MDSC alters the expression of genes related to cell proliferation, neural growth factor and extracellular matrix and the expression of smooth and striated muscle proteins in traumatized rat urethra.
id UFSP_65369d1249e5f3d1fab148f1f1f8426a
oai_identifier_str oai:repositorio.unifesp.br/:11600/49880
network_acronym_str UFSP
network_name_str Repositório Institucional da UNIFESP
repository_id_str 3465
spelling Avaliação molecular e imunohistoquímica da uretra de ratas após trauma induzido e terapia intravenosa com células-tronco derivadas de músculoMolecular and immunohistochemical evaluation of urethra of female rats after induced trauma and intravenous therapy with muscle derived stem cellsStem cellsUrinary incontinenceUrethraIncontinência urináriaCélulas roncoUretraObjective: To identify the migration to the urethra of muscle derived stem cells (MDSCs) after intravenous (IV) injection in rats submitted to pelvic floor trauma due to vaginal distension (DV) and to analyze the effects of MDSC in the urethra of rats after seven and thirty days of the trauma in regards to: (1) mRNA expression of collagens, vascular endothelial growth factor (VEGF), nerve growth factor (NGF), Ki67 cell proliferation marker, and the expression of genes related to smooth and striated muscle apparatus; (2) expression of smooth and striated muscle proteins. Methods: MDSCs expressing green fluorescent protein (GFP) were injected into the tail vein of the rats at day three after DV. The urethras of five groups were analyzed: Control, Trauma 7 days (animals submitted to DV, sacrificed 7 days), MDSC 7 days (animals submitted to DV receiving IV therapy with MDSC, sacrificed 7 days), Trauma 30 days (animals submitted to DV, sacrificed at 30 days), MDSC 30 days (animals submitted to DV receiving IV therapy with MDSC, sacrificed at 30 days). Location of GFP cells was verified at 2 hours and 7 days after IV injection. RT-qPCR was used for gene expression and immunohistochemistry for protein identification and quantification. Results: MDSCs were early identified in the urethra, but only deformed cells after 7 days. After seven days of trauma, the MYH11 gene was overexpressed in the Trauma group in relation to Control (p=0.01). Expression of the mKi67 gene was increased in the MDSC group relative to Trauma (p=0.03) and Control (p=0.02). Expression of the COL1A1 and COL3A1 genes increased in the MDSC group relative to the Control (p=0.001 and p=0.002). The gene expression of NGF was decreased in the MDSC group in relation to Trauma (p=0.001) and there was no difference in expression of the MYH2, Desmin and VEGF gene. In the same period, immunohistochemical expression of MHY11, MYH2 and Desmin was increased in the MDSC group in relation to Trauma (p=0.01, p=0.005 and p=0.02) and decreased in the Trauma group in relation to the Control (p =0.01, p=0.008 and p=0.03). After 30 days of trauma, MYH11 and MYH2 gene expression was increased in the Trauma group compared to Control (p=0.03 and p=0.04). Expression of the mKi67 gene was increased in the MDSC group relative to Trauma (p=0.02) and Control (p=0.01). Expression of the COL1A1 gene increased in the MDSC group compared to the Control (p=0.008) and Trauma (p=0.03) and COL3A1 gene expression increased in the MDSC group compared to Control (p=0.03). However, the gene expression of NGF was decreased in the MDSC group in relation to Trauma (p=0.002) and there was no difference in the gene expression of Desmin and VEGF. In the same period, immunohistochemical expression of MYH11, MYH2 and Desmin was increased in the MDSC group in relation to Trauma (p=0.01, p=0.002 and p=0.01) and MYH2 gene expression decreased in the Trauma group compared to Control (p=0.04). Conclusion: MDSCs migrated early to the traumatized urethra, but did not integrate into the tissue. IV administration of MDSC alters the expression of genes related to cell proliferation, neural growth factor and extracellular matrix and the expression of smooth and striated muscle proteins in traumatized rat urethra.Objetivo: Identificar a migração para uretra decélulas-tronco derivadas de músculo (MDSCs) após injeção intravenosa (IV) em ratas submetidas ao trauma do assoalho pélvico por distensão vaginal (DV). Analisar o efeito da injeção IV de MDSC, sete e trinta dias após DV através da expressão dos genes miosina de cadeia pesada do músculo liso (MYH11), miosina de cadeia pesada do músculo esquelético (MYH2), Desmina, marcador de proliferação celular mKi67, colágeno tipo I (COL1A1), colágeno tipo III (COL3A1), fator de crescimento neural (NGF) e fator de crescimento endotelial vascular A (VEGFa) e da expressão imunohistoquímica das proteínas MYH11, MYH2 e Desmina.Métodos: MDSCs que expressavam proteína fluorescente verde (GFP) foram injetadas na veia da cauda das ratas três dias após DV. Foram analisadas uretras de cinco grupos: Controle, Trauma 7 dias (animais submetidos a DV, sacrificados com 7 dias), MDSC 7 dias (animais submetidos a DV que receberam terapia IV com MDSC, sacrificados com 7 dias), Trauma 30 dias (animais submetidos a DV, sacrificados com 30 dias), MDSC 30 dias (animais submetidos a DV que receberam terapia IV com MDSC, sacrificados com 30 dias). Foi realizada imunofluorescência para localização das MDSCs com 2 horas e 7 dias após injeção IV.RT-qPCR foi utilizado para expressão gênica e imunohistoquímica para identificação e quantificação proteica.Resultados:Foi identificada presença precoce das MDSCs na uretra, não sendo observadas após 7 dias. Após sete dias do trauma, o gene MYH11 foi sobre-expresso no grupo Trauma em relação ao Controle (p=0,01). Expressão do gene mKi67 estava aumentada no grupo MDSC em relação ao Trauma (p=0,03) e Controle (p=0,02).Expressão dos genes COL1A1 e COL3A1 aumentou no grupo MDSC em relação ao Controle (p=0,001 e p=0,002). Já a expressão gênica do NGF foi diminuída no grupo MDSC em relação ao Trauma (p=0,001) e não houve diferença na expressão do gene MYH2, Desmina e VEGF. Nesse mesmo período, expressão imunohistoquímica de MYH11, MYH2 e Desmina estava aumentada no grupo MDSC em relação ao Trauma (p=0,01; p=0,005 e p=0,02) e diminuída no grupo Trauma em relação ao Controle (p=0,01; p=0,008 e p=0,03).Após 30 dias do trauma, expressão dos genes MYH11 e MYH2 foi aumentada no grupo Trauma em relação ao Controle (p=0,03 e p=0,04). Expressão do gene mKi67 foi aumentada no grupo MDSC em relação ao Trauma (p=0,02) e Controle (p=0,01). Expressão do gene COL1A1 aumentou no grupo MDSC em relação aos grupos Controle (p=0,008) e Trauma (p=0,03) e expressão do gene COL3A1 aumentou no grupo MDSC em comparação ao Controle (p=0,03). Já a expressão gênica do NGF foi diminuída no grupo MDSC em relação ao Trauma (p=0,002) e não houve diferença na expressão gênica da Desmina e VEGF. Nesse mesmo período, expressão imunohistoquímica de MYH11, MYH2 e Desmina estava aumentada no grupo MDSC em relação ao Trauma (p=0,01; p=0,002 e p=0,01) e expressão do gene MYH2 diminuída no grupo Trauma em relação ao Controle (p=0,04).Conclusão:MDSCs migraram precocemente para uretra traumatizada, mas não se integraram ao tecido. Administração IV de MDSC altera a expressão de genes relacionados à proliferação celular, fator de crescimento neural e matriz extracelular e a expressão de proteínas da musculatura lisa e estriada em uretra traumatizada de ratas.Dados abertos - Sucupira - Teses e dissertações (2017)Universidade Federal de São Paulo (UNIFESP)Castro, Rodrigo de Aquino [UNIFESP]Bortolini, Maria Augusta Tezelli [UNIFESP]http://lattes.cnpq.br/1150368284144393http://lattes.cnpq.br/6590913930590292http://lattes.cnpq.br/8795441347109037Universidade Federal de São Paulo (UNIFESP)Bilhar, Andreisa Paiva Monteiro [UNIFESP]2019-06-19T14:57:02Z2019-06-19T14:57:02Z2017-02-22info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersion108 f.application/pdfhttps://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=5760899http://repositorio.unifesp.br/handle/11600/49880porSão Pauloinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP2024-08-10T09:51:40Zoai:repositorio.unifesp.br/:11600/49880Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestbiblioteca.csp@unifesp.bropendoar:34652024-08-10T09:51:40Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false
dc.title.none.fl_str_mv Avaliação molecular e imunohistoquímica da uretra de ratas após trauma induzido e terapia intravenosa com células-tronco derivadas de músculo
Molecular and immunohistochemical evaluation of urethra of female rats after induced trauma and intravenous therapy with muscle derived stem cells
title Avaliação molecular e imunohistoquímica da uretra de ratas após trauma induzido e terapia intravenosa com células-tronco derivadas de músculo
spellingShingle Avaliação molecular e imunohistoquímica da uretra de ratas após trauma induzido e terapia intravenosa com células-tronco derivadas de músculo
Bilhar, Andreisa Paiva Monteiro [UNIFESP]
Stem cells
Urinary incontinence
Urethra
Incontinência urinária
Células ronco
Uretra
title_short Avaliação molecular e imunohistoquímica da uretra de ratas após trauma induzido e terapia intravenosa com células-tronco derivadas de músculo
title_full Avaliação molecular e imunohistoquímica da uretra de ratas após trauma induzido e terapia intravenosa com células-tronco derivadas de músculo
title_fullStr Avaliação molecular e imunohistoquímica da uretra de ratas após trauma induzido e terapia intravenosa com células-tronco derivadas de músculo
title_full_unstemmed Avaliação molecular e imunohistoquímica da uretra de ratas após trauma induzido e terapia intravenosa com células-tronco derivadas de músculo
title_sort Avaliação molecular e imunohistoquímica da uretra de ratas após trauma induzido e terapia intravenosa com células-tronco derivadas de músculo
author Bilhar, Andreisa Paiva Monteiro [UNIFESP]
author_facet Bilhar, Andreisa Paiva Monteiro [UNIFESP]
author_role author
dc.contributor.none.fl_str_mv Castro, Rodrigo de Aquino [UNIFESP]
Bortolini, Maria Augusta Tezelli [UNIFESP]
http://lattes.cnpq.br/1150368284144393
http://lattes.cnpq.br/6590913930590292
http://lattes.cnpq.br/8795441347109037
Universidade Federal de São Paulo (UNIFESP)
dc.contributor.author.fl_str_mv Bilhar, Andreisa Paiva Monteiro [UNIFESP]
dc.subject.por.fl_str_mv Stem cells
Urinary incontinence
Urethra
Incontinência urinária
Células ronco
Uretra
topic Stem cells
Urinary incontinence
Urethra
Incontinência urinária
Células ronco
Uretra
description Objective: To identify the migration to the urethra of muscle derived stem cells (MDSCs) after intravenous (IV) injection in rats submitted to pelvic floor trauma due to vaginal distension (DV) and to analyze the effects of MDSC in the urethra of rats after seven and thirty days of the trauma in regards to: (1) mRNA expression of collagens, vascular endothelial growth factor (VEGF), nerve growth factor (NGF), Ki67 cell proliferation marker, and the expression of genes related to smooth and striated muscle apparatus; (2) expression of smooth and striated muscle proteins. Methods: MDSCs expressing green fluorescent protein (GFP) were injected into the tail vein of the rats at day three after DV. The urethras of five groups were analyzed: Control, Trauma 7 days (animals submitted to DV, sacrificed 7 days), MDSC 7 days (animals submitted to DV receiving IV therapy with MDSC, sacrificed 7 days), Trauma 30 days (animals submitted to DV, sacrificed at 30 days), MDSC 30 days (animals submitted to DV receiving IV therapy with MDSC, sacrificed at 30 days). Location of GFP cells was verified at 2 hours and 7 days after IV injection. RT-qPCR was used for gene expression and immunohistochemistry for protein identification and quantification. Results: MDSCs were early identified in the urethra, but only deformed cells after 7 days. After seven days of trauma, the MYH11 gene was overexpressed in the Trauma group in relation to Control (p=0.01). Expression of the mKi67 gene was increased in the MDSC group relative to Trauma (p=0.03) and Control (p=0.02). Expression of the COL1A1 and COL3A1 genes increased in the MDSC group relative to the Control (p=0.001 and p=0.002). The gene expression of NGF was decreased in the MDSC group in relation to Trauma (p=0.001) and there was no difference in expression of the MYH2, Desmin and VEGF gene. In the same period, immunohistochemical expression of MHY11, MYH2 and Desmin was increased in the MDSC group in relation to Trauma (p=0.01, p=0.005 and p=0.02) and decreased in the Trauma group in relation to the Control (p =0.01, p=0.008 and p=0.03). After 30 days of trauma, MYH11 and MYH2 gene expression was increased in the Trauma group compared to Control (p=0.03 and p=0.04). Expression of the mKi67 gene was increased in the MDSC group relative to Trauma (p=0.02) and Control (p=0.01). Expression of the COL1A1 gene increased in the MDSC group compared to the Control (p=0.008) and Trauma (p=0.03) and COL3A1 gene expression increased in the MDSC group compared to Control (p=0.03). However, the gene expression of NGF was decreased in the MDSC group in relation to Trauma (p=0.002) and there was no difference in the gene expression of Desmin and VEGF. In the same period, immunohistochemical expression of MYH11, MYH2 and Desmin was increased in the MDSC group in relation to Trauma (p=0.01, p=0.002 and p=0.01) and MYH2 gene expression decreased in the Trauma group compared to Control (p=0.04). Conclusion: MDSCs migrated early to the traumatized urethra, but did not integrate into the tissue. IV administration of MDSC alters the expression of genes related to cell proliferation, neural growth factor and extracellular matrix and the expression of smooth and striated muscle proteins in traumatized rat urethra.
publishDate 2017
dc.date.none.fl_str_mv 2017-02-22
2019-06-19T14:57:02Z
2019-06-19T14:57:02Z
dc.type.driver.fl_str_mv info:eu-repo/semantics/doctoralThesis
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format doctoralThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=5760899
http://repositorio.unifesp.br/handle/11600/49880
url https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=5760899
http://repositorio.unifesp.br/handle/11600/49880
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 108 f.
application/pdf
dc.coverage.none.fl_str_mv São Paulo
dc.publisher.none.fl_str_mv Universidade Federal de São Paulo (UNIFESP)
publisher.none.fl_str_mv Universidade Federal de São Paulo (UNIFESP)
dc.source.none.fl_str_mv reponame:Repositório Institucional da UNIFESP
instname:Universidade Federal de São Paulo (UNIFESP)
instacron:UNIFESP
instname_str Universidade Federal de São Paulo (UNIFESP)
instacron_str UNIFESP
institution UNIFESP
reponame_str Repositório Institucional da UNIFESP
collection Repositório Institucional da UNIFESP
repository.name.fl_str_mv Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)
repository.mail.fl_str_mv biblioteca.csp@unifesp.br
_version_ 1814268344923062272