Estudo de vesículas extracelulares produzidas por cardiomiócitos humanos, derivados de células-tronco pluripotentes, em homeostase e estresse celular

• Ano de defesa: 2019
• Autor(a) principal: Paiva, Dayane Silva de
• Orientador(a): Carvalho, Juliana Lott de
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Católica de Brasília
• Programa de Pós-Graduação: Programa Stricto Sensu em Ciências Genômicas e Biotecnologia
• Departamento: Escola de Saúde e Medicina
• País: Brasil
• Palavras-chave em Português: Doxorrubicina; Células-tronco; Cardiomiócitos; Toxicologia in vitro; Vesículas extracelulares; Doenças cardiovasculares
• Palavras-chave em Inglês: Cardiovascular disease; Extracellular vesicles; In vitro toxicology; Cardiomyocyte; Doxorubicin; Stem cell
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::GENETICA
• Resumo em Inglês: Hundreds thousands of people die every year in Brazil and the world due to heart diseases. The possible causes for the development of these pathologies are: due to unhealthy eating habits, exposure to other agents considered to be toxic to heart health, as an example. Some of these toxic agents could be drugs with side effects such as antitumor medicines. Doxorubicin is a drug widely used in the treatment of several solid tumors. However, it has a high cardiovascular side effect, causing hypertrophy. The cardiac cells can be obtained from stem cells (iPSCs) which are an important source of obtaining tissue-specific cells. The present work focused on obtaining cardiomyocytes derived from iPSCs to study the extracellular vesicles (EV's) in homeostasis and stress by Doxorubicin (DOXO). The differentiation of cardiomyocytes was successful and the cells characterization showed expression of specific markers such as MIXL1, GATA4, hTNNT, immunofluorescence positive for cardiac troponin and spontaneous cellular contraction. The MTT assay performed at the concentrations of 0.1 μM and 0.5 μM of DOXO showed that the concentration of 0.1 μM is ideal for the following assays. The RT-PCR evaluation demonstrated reduced expression of BCL2, TGβ1 and p53 genes in DOXO-treated cardiomyocytes and increased FGF2 expression, consistent with the profile of DOXO-induced hypertrophic and cardiac pathologies. The isolation of the extracellular vesicles produced by cardiomyocytes by the different methods did not present a great difference by Bradford assay quantification. RT-PCR performed on isolated EV’s transcripts did not show cell-like expression profile. BCL2 and FGF2 genes showed increased expression of the EV’s isolated from stressed cardiomyocytes compared to the control and the TGFβ1 and p53 genes had the reduced level of expression in EV’s of stressed cardiomyocytes. Showing that could be a selection of intentional transcripts for cellular communication. The data obtained suggest that the mRNA content of the cells and derived EV’s may be diverse and additional studies on the stress by DOXO would be enlightening in the understanding pathophysiology of this intoxication.
• Link de acesso: https://bdtd.ucb.br:8443/jspui/handle/tede/2557

Hundreds thousands of people die every year in Brazil and the world due to heart diseases. The possible causes for the development of these pathologies are: due to unhealthy eating habits, exposure to other agents considered to be toxic to heart health, as an example. Some of these toxic agents could be drugs with side effects such as antitumor medicines. Doxorubicin is a drug widely used in the treatment of several solid tumors. However, it has a high cardiovascular side effect, causing hypertrophy. The cardiac cells can be obtained from stem cells (iPSCs) which are an important source of obtaining tissue-specific cells. The present work focused on obtaining cardiomyocytes derived from iPSCs to study the extracellular vesicles (EV's) in homeostasis and stress by Doxorubicin (DOXO). The differentiation of cardiomyocytes was successful and the cells characterization showed expression of specific markers such as MIXL1, GATA4, hTNNT, immunofluorescence positive for cardiac troponin and spontaneous cellular contraction. The MTT assay performed at the concentrations of 0.1 μM and 0.5 μM of DOXO showed that the concentration of 0.1 μM is ideal for the following assays. The RT-PCR evaluation demonstrated reduced expression of BCL2, TGβ1 and p53 genes in DOXO-treated cardiomyocytes and increased FGF2 expression, consistent with the profile of DOXO-induced hypertrophic and cardiac pathologies. The isolation of the extracellular vesicles produced by cardiomyocytes by the different methods did not present a great difference by Bradford assay quantification. RT-PCR performed on isolated EV’s transcripts did not show cell-like expression profile. BCL2 and FGF2 genes showed increased expression of the EV’s isolated from stressed cardiomyocytes compared to the control and the TGFβ1 and p53 genes had the reduced level of expression in EV’s of stressed cardiomyocytes. Showing that could be a selection of intentional transcripts for cellular communication. The data obtained suggest that the mRNA content of the cells and derived EV’s may be diverse and additional studies on the stress by DOXO would be enlightening in the understanding pathophysiology of this intoxication.