Modelo de pele humana reconstru??da como plataforma para estudos de fotoenvelhecimento

Autor: Almeida, Bianca de Lima
Přispěvatelé: Vasconcellos, Marne Carvalho de, Montenegro, Raquel Carvalho, Lima, Emerson Silva
Jazyk: portugalština
Rok vydání: 2020
Předmět:
Zdroj: Biblioteca Digital de Teses e Dissertações da UFAM
Universidade Federal do Amazonas (UFAM)
instacron:UFAM
Popis: Submitted by Bianca Almeida (bia0107@hotmail.com) on 2020-07-20T18:42:21Z No. of bitstreams: 4 Disserta????o formatada Bianca 20.07.2020.pdf: 1860033 bytes, checksum: 610fddf72b019dad98fcb5146261a8d4 (MD5) CartaEncaminhamentoAutodepo??sito2.pdf: 54664 bytes, checksum: 03e864494f39c0bd5886ea8d5a84b7f9 (MD5) Folha de aprova????o bianca.pdf: 346517 bytes, checksum: 206ab5df14a343f5747e1b00e81b715f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Approved for entry into archive by PPGCF Ci??ncias Farmac??uticas (ppgcf@ufam.edu.br) on 2020-07-22T13:33:17Z (GMT) No. of bitstreams: 4 Disserta????o formatada Bianca 20.07.2020.pdf: 1860033 bytes, checksum: 610fddf72b019dad98fcb5146261a8d4 (MD5) CartaEncaminhamentoAutodepo??sito2.pdf: 54664 bytes, checksum: 03e864494f39c0bd5886ea8d5a84b7f9 (MD5) Folha de aprova????o bianca.pdf: 346517 bytes, checksum: 206ab5df14a343f5747e1b00e81b715f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Approved for entry into archive by Divis??o de Documenta????o/BC Biblioteca Central (ddbc@ufam.edu.br) on 2020-07-23T14:21:13Z (GMT) No. of bitstreams: 4 Disserta????o formatada Bianca 20.07.2020.pdf: 1860033 bytes, checksum: 610fddf72b019dad98fcb5146261a8d4 (MD5) CartaEncaminhamentoAutodepo??sito2.pdf: 54664 bytes, checksum: 03e864494f39c0bd5886ea8d5a84b7f9 (MD5) Folha de aprova????o bianca.pdf: 346517 bytes, checksum: 206ab5df14a343f5747e1b00e81b715f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Made available in DSpace on 2020-07-23T14:21:13Z (GMT). No. of bitstreams: 4 Disserta????o formatada Bianca 20.07.2020.pdf: 1860033 bytes, checksum: 610fddf72b019dad98fcb5146261a8d4 (MD5) CartaEncaminhamentoAutodepo??sito2.pdf: 54664 bytes, checksum: 03e864494f39c0bd5886ea8d5a84b7f9 (MD5) Folha de aprova????o bianca.pdf: 346517 bytes, checksum: 206ab5df14a343f5747e1b00e81b715f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2020-01-30 CAPES - Coordena????o de Aperfei??oamento de Pessoal de N??vel Superior The effects of exposure to ultraviolet radiation on human skin have aroused interest in the scientific class, in understanding the transformations involved. Models of human skin reconstructed in vitro were developed as alternative methods to the use of animals in experimentation and proved to be useful and effective tools for studies. These models express different receptors, have proliferative capacity, extracellular matrix synthesis and metabolic functions, becoming tools of choice to understand the biological mechanisms of the skin. The validated and commercially available models are reconstructed from the cultivation of primary cells, facing some limitations in terms of acquisition and reproduction, which can affect their applicability. This work aimed to standardize a model of human skin reconstructed from permanent cell lines and evaluate the effects of Ultraviolet Radiation on this model. To this end, the work consisted of standardizing the model developed with permanent culture human keratinocytes and fibroblasts on a transwell-type membrane support for subsequent exposure to ultraviolet B radiation. For standardization, the cultivation times 10, 15 and 18 days were tested. Cell viability assays were performed using the MTT method and histological analysis by staining with hematoxylin and eosin. The condition that best represented the morphological structure of human skin, with epidermis and dermis, was 10 days of culture. The radiation doses used were 50mJ / cm2, 150mJ / cm2, 500mJ / cm2 and 1500mJ / cm2. At the lowest doses there was a small reduction in cell viability (50 and 150 mJ / cm2), the opposite occurred at doses of 500 and 1500mJ / cm2, showing a viability reduction of more than 50%. Histological sections showed a change in the epidermal layer of the skin after exposure, in a dose-dependent manner, visualizing the decrease in epithelial thickness, separation and vacuolization of cells in the epithelial layer. The proteolytic activity of MMPs 2 and 9 was assessed using a zymographic method. The lowest radiation dose triggered confirmed genotoxic activity, through the comet assay in alkaline pH. In view of the above, with this work it was possible to standardize a model of human skin reconstructed in vitro using lineage of permanent cells as a platform for photoaging studies using UVB radiation. Future experiments are needed to assess the potential for radiation damage and to elucidate the mechanisms involved, as well as the behavior of cells exposed in the three-dimensional microenvironment. Os efeitos da exposi????o ?? radia????o ultravioleta na pele humana t??m despertado interesse na classe cient??fica, no entendimento das transforma????es envolvidas. Modelos de pele humana reconstru??da in vitro foram desenvolvidos como m??todos alternativos ?? utiliza????o de animais em experimenta????o e mostraram ser ferramentas ??teis e eficazes para estudos. Esses modelos expressam diferentes receptores, possuem capacidade proliferativa, s??ntese de matriz extracelular e fun????es metab??licas, tornando-se ferramentas de escolha para entender os mecanismos biol??gicos da pele. Os modelos validados e comercialmente dispon??veis s??o reconstru??dos a partir do cultivo de c??lulas prim??rias, enfrentando algumas limita????es, quanto a aquisi????o e reprodu????o, podendo afetar sua aplicabilidade. Este trabalho teve objetivo de padronizar um modelo de pele humana reconstru??da a partir de linhagens de c??lulas permanentes e avaliar os efeitos da Radia????o Ultravioleta B nesse modelo. Para isso, o trabalho consistiu na padroniza????o do modelo desenvolvido com queratin??citos e fibroblastos humanos de cultivo permanente em um suporte de membrana do tipo transwell para posterior exposi????o ?? radia????o ultravioleta B. Para a padroniza????o, foram testados os tempos de cultivo 10, 15 e 18 dias. Os ensaios de viabilidade celular foram realizados pelo m??todo do MTT e a an??lise histol??gica por colora????o com hematoxilina e eosina. A condi????o que melhor representou a estrutura morfol??gica da pele humana, com epiderme e derme foi a de 10 dias de cultivo. As doses de radia????o utilizadas foram 50mJ/cm2, 150mJ/cm2, 500mJ/cm2 e 1500mJ/cm2. Nas menores doses houve uma pequena redu????o na viabilidade celular (50 e 150 mJ/cm2), o oposto ocorreu nas doses de 500 e 1500mJ/cm2, apresentando redu????o da viabilidade em mais de 50%. Os cortes histol??gicos evidenciaram altera????o na camada epid??rmica da pele ap??s exposi????o, de forma dose-dependente, visualizando a diminui????o da espessura do epit??lio, separa????o e vacuoliza????o das c??lulas na camada epitelial. A atividade proteol??tica de MMPs 2 e 9 foi avaliada por m??todo zimogr??fico. A menor dose de radia????o desencadeou atividade genot??xica confirmada, por meio do ensaio cometa em pH alcalino. Diante do exposto, com este trabalho foi poss??vel padronizar um modelo de pele humana reconstrui??da in vitro utilizando linhagem de c??lulas permanentes como plataforma de estudos de fotoenvelhecimento por meio de radia????o UVB. Experimentos futuros s??o necess??rios para avaliar o potencial dos danos causados pela radia????o e elucidar os mecanismos envolvidos, bem como o comportamento das c??lulas expostas no microambiente tridimensional.
Databáze: OpenAIRE
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