Caracterização de célula progenitora hepática no cultivo celular de fígado de feto canino
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Data
2016-02-25
Autores
Tavares, Mariana Riboli [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Resumo - Com a descoberta das células-tronco abriu-se um leque de possibilidades nas pesquisas de nichos destas em diferentes órgãos e também no estudo de doenças e regeneração tecidual. O fígado vem sendo alvo de muitos estudos já que as doenças crônicas deste órgão tem como resolução um transplante, o que é caro e há poucos doadores. O fígado é composto, principalmente, por hepatócitos, no entanto, seu cultivo e mantença in vitro são difíceis. Assim, uma alternativa é a manutenção das células progenitoras hepáticas. Elas são conhecidas em camundongos, humanos, mas, pouco mencionadas em cães. Esse trabalho objetivou mostrar a existência das células progenitoras hepáticas no cultivo celular de fígado fetal de cães por imunocitoquímica e citometria de fluxo a fim de saber se este nicho celular existe no cultivo e se é possível num futuro próximo a separação desta linhagem nesta espécie. Foram utilizados para a realização do trabalho 10 fetos sendo que destes foi possível o aproveitamento em sete animais. Foi realizado no trabalho isolamento, cultivo de células hepáticas, criopreservação, curva de crescimento celular, imnocitoquímica e citometria de fluxo. A cultura celular durou sete passagens, com dobramento populacional de 31,7 horas, na imunocitoquímica as células foram marcadas positivamente para os seguintes marcadores: EpCAM, NCAM, Nestina e Thy-1.Na citometria de fluxo foi possível realizar a dupla marcação das células com os maracadores EpCam e NCAM que resultaram em 0,5% ±0,173 do total de células analisadas, conclui-se que o objetivo da pesquisa almejado foi conseguido, existe células progenitoras hepáticas no cultivo celular de fígado fetal canino.
Abstract - With the discovery of stem cells has opened up a range of possibilities in niche research these in different organs and also in the study of diseases and tissue regeneration the liver has been the subject of many studies since chronic diseases of this organ has the resolution a transplant, which is expensive and there are few donors. The liver consists mainly of hepatocytes; however, its cultivation and maintenance in vitro are difficult. Thus, an alternative is to maintain the hepatic progenitor cells. They are known in mice, humans, but rarely mentioned in dogs. This study aimed to show the existence of hepatic progenitor cells in cell culture fetal liver of dogs by immunocytochemistry and flow cytometry in order to know if this cell niche exists in the cultivation and whether it is possible in the near future the separation of this lineage in this species. They were used to carry out the work 10 fetuses and of these it was possible to use in seven animals. Work was carried out in isolation, cultivation of liver cells, cryopreservation, cell growth curve, immunocytochemistry and flow cytometry. The cell culture lasted seven passes, doubling time in 31.7 hours, in immunocytochemistry cells were positively stained for the following markers: EpCAM, NCAM, Nestin and Thy-1. In flow cytometry it was possible to double staining of cells with EpCam markers and NCAM which resulted in 0.5 % ± 0.173 of the total of the cells analyzed. Concludes that the purpose of the desired search has been achieved there liver progenitor cells in fetal liver cell culture canine
Abstract - With the discovery of stem cells has opened up a range of possibilities in niche research these in different organs and also in the study of diseases and tissue regeneration the liver has been the subject of many studies since chronic diseases of this organ has the resolution a transplant, which is expensive and there are few donors. The liver consists mainly of hepatocytes; however, its cultivation and maintenance in vitro are difficult. Thus, an alternative is to maintain the hepatic progenitor cells. They are known in mice, humans, but rarely mentioned in dogs. This study aimed to show the existence of hepatic progenitor cells in cell culture fetal liver of dogs by immunocytochemistry and flow cytometry in order to know if this cell niche exists in the cultivation and whether it is possible in the near future the separation of this lineage in this species. They were used to carry out the work 10 fetuses and of these it was possible to use in seven animals. Work was carried out in isolation, cultivation of liver cells, cryopreservation, cell growth curve, immunocytochemistry and flow cytometry. The cell culture lasted seven passes, doubling time in 31.7 hours, in immunocytochemistry cells were positively stained for the following markers: EpCAM, NCAM, Nestin and Thy-1. In flow cytometry it was possible to double staining of cells with EpCam markers and NCAM which resulted in 0.5 % ± 0.173 of the total of the cells analyzed. Concludes that the purpose of the desired search has been achieved there liver progenitor cells in fetal liver cell culture canine
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Palavras-chave
EpCAM, NCAM, Célula-tronco hepática, Cão, EpCAM, NCAM, Stem cells hepatic, Dog