Transcriptoma e caracterização do perfil proteômico de células-tronco mesenquimais do tecido adiposo de mini-horses (equus ferus caballus)
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Data
2019-09-30
Autores
Maciel, Alice Pereira
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Universidade Estadual Paulista (Unesp)
Resumo
As células-tronco mesenquimais extraídas da medula óssea ou tecido adiposo são utilizadas em abordagens terapêuticas, em vista das suas propriedades anti-inflamatórias, imunomoduladoras e regenerativas. Estas células são consideradas uma nova alternativa para o tratamento de enfermidades em diversas espécies, incluindo as dos equinos. Dessa forma, com o intuito de investigar a possível utilização das células estromais de mini-horses em tratamentos de cavalos, nesse estudo foi realizada a colheita, isolamento, expansão, caracterização imunofenotípica e diferenciação em duas linhagens mesenquimais: osteogênica e adipogênica de células estromais de mini-horses (Equus ferus caballus), objetivando a formação de um banco celular. Foi ainda realizada a análise transcricional comparativa dessas células, com as células estromais oriundas de cavalos (Equus caballus), produzidas e caracterizadas nas mesmas condições. Os parâmetros analisados foram o perfil de genes relacionados à angiogênese, regulação negativa da proliferação de células T, regulação negativa da ativação de células T, proliferação negativa de linfócitos reguladores, regulação negativa da proliferação de células mononucleares, regulação negativa da adesão célula-célula de leucócitos, regulação negativa da proliferação de leucócitos, regulação negativa da adesão célula-leucócito, regulação negativa da ativação de linfócitos. Também foram pesquisados os principais genes up e down regulados comparando mini-horses aos cavalos. Para formação do banco celular foram realizadas avaliações que comprovaram as características de células estromais nas células obtidas dos mini-horses, a avaliação imunofenotípica por citometria de fluxo demonstrou expressão alta dos marcadores CD90 (99,83 ± 0,28) e esperada do CD105 (38,66 ± 18,47) e expressão baixa dos marcadores CD34 (3,55 ± 1,14) e MHC II (4,21 ± 2,03). O potencial de diferenciação em linhagens adipogênicas in vitro foi observado após o 14º dia através da visualização da deposição de gotículas lipídicas no citoplasma, e a linhagem osteogênica foi identificada após o 21° dia pela coloração positiva da matriz de cálcio extracelular. Ao final da avaliação transcricional, observamos um perfil de expressão gênica diferente entre os grupos, sugerindo que as diferenças entre os animais estudados se expande a mutação do gene Aggrecan que determina o nanismo. Apesar disso, os resultados indicam que as células estromais de mini-horse podem ser utilizadas como uma alternativa para o tratamento de lesões equinas com a vantagem de apresentarem uma maior expressão de genes relacionados à imunomodulação.
Mesenchymal stem cells extracted from bone marrow or adipose tissue are used in therapeutic approaches in view of their anti-inflammatory, immunomodulatory and regenerative properties. These cells are considered a new alternative for the treatment of diseases in several species. In order to investigate the possible use of mini-horses stromal cells in horse treatments, in this study was performed the collection, isolation, expansion, immunophenotypic characterization and differentiation in two mesenchymal lines were performed: osteogenic and adipogenic stromal cell mini-horses (Equus ferus caballus), aiming the formation of a cellular bank. We also performed the comparative transcriptional analysis of these cells, with stromal cells from horses (Equus caballus), produced and characterized under the same conditions. The parameters analyzed were the profile of genes related to angiogenesis, negative regulation of T cell proliferation, negative regulation of T cell activation, negative proliferation of regulatory lymphocytes, negative regulation of mononuclear cell proliferation, negative regulation of cell-cell adhesion leukocytes, negative regulation of leukocyte proliferation, negative regulation of cell-leukocyte adhesion, negative regulation of lymphocyte activation. We also searched the top 10 genes up and down regulated comparing mini-horses to horses. For cell bank formation tests were performed that demonstrated the characteristics of stromal cells in the cells obtained from the mini-horses, the immunophenotypic evaluation by flow cytometry showed high CD90 (99.83 ± 0.28) and expected CD105 ( 38.66 ± 18.47) and low expression of the CD34 (3.55 ± 1.14) and MHC II (4.21 ± 2.03) markers. The potential for differentiation in in vitro adipogenic lines was observed after day 14 by visualization of the deposition of lipid droplets in the cytoplasm, and the osteogenic lineage was identified after 21 ° day by the positive staining of the extracellular calcium matrix. At the end of the transcriptional evaluation, we observed a different gene expression profile between the groups, suggesting that the differences between the studied animals expands the Aggrecan gene mutation that determines the dwarfism. However, the results strongly indicate that ASCs from mini-horses can be used as an alternative to treat horse’s diseases, with the vantages of presenting a higher expression of immunomodulatory genes
Mesenchymal stem cells extracted from bone marrow or adipose tissue are used in therapeutic approaches in view of their anti-inflammatory, immunomodulatory and regenerative properties. These cells are considered a new alternative for the treatment of diseases in several species. In order to investigate the possible use of mini-horses stromal cells in horse treatments, in this study was performed the collection, isolation, expansion, immunophenotypic characterization and differentiation in two mesenchymal lines were performed: osteogenic and adipogenic stromal cell mini-horses (Equus ferus caballus), aiming the formation of a cellular bank. We also performed the comparative transcriptional analysis of these cells, with stromal cells from horses (Equus caballus), produced and characterized under the same conditions. The parameters analyzed were the profile of genes related to angiogenesis, negative regulation of T cell proliferation, negative regulation of T cell activation, negative proliferation of regulatory lymphocytes, negative regulation of mononuclear cell proliferation, negative regulation of cell-cell adhesion leukocytes, negative regulation of leukocyte proliferation, negative regulation of cell-leukocyte adhesion, negative regulation of lymphocyte activation. We also searched the top 10 genes up and down regulated comparing mini-horses to horses. For cell bank formation tests were performed that demonstrated the characteristics of stromal cells in the cells obtained from the mini-horses, the immunophenotypic evaluation by flow cytometry showed high CD90 (99.83 ± 0.28) and expected CD105 ( 38.66 ± 18.47) and low expression of the CD34 (3.55 ± 1.14) and MHC II (4.21 ± 2.03) markers. The potential for differentiation in in vitro adipogenic lines was observed after day 14 by visualization of the deposition of lipid droplets in the cytoplasm, and the osteogenic lineage was identified after 21 ° day by the positive staining of the extracellular calcium matrix. At the end of the transcriptional evaluation, we observed a different gene expression profile between the groups, suggesting that the differences between the studied animals expands the Aggrecan gene mutation that determines the dwarfism. However, the results strongly indicate that ASCs from mini-horses can be used as an alternative to treat horse’s diseases, with the vantages of presenting a higher expression of immunomodulatory genes
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Palavras-chave
cultivo celular, diferenciação, equinos, gene, terapia celular, cell culture, cell therapy, differentiation, equines,