Indução de diferenciação causa mudança na localização subcelular de Runx em células-tronco neurais
Data
2015-04-29
Autores
Massinhani, Fernando Henrique 
Orientadores
Porcionatto, Marimelia Aparecida
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Os membros da família Runx são fatores de transcrição, evolutivamente conservados, que regulam a expressão de genes envolvidos na diferenciação e na progressão do ciclo celular, podendo atuar como ativadores ou repressores transcricionais. Assim, o objetivo desta tese foi verificar se a expressão dos membros da família Runx (Runx1, 2 e 3) por células-tronco neurais da zona subventricular (SVZ) de camundongos adultos está relacionada com a diferenciação dessas células em neurônios. Inicialmente, observamos a expressão gênica e proteica dos membros da família Runx por PCR e Western blotting respectivamente, utilizando células-tronco neurais cultivadas como neurosferas. Em seguida, realizamos imunocitoquímica com neurosferas mantidas em cultura por 1, 7 e 14 dias após a adesão ao substrato, para promover a diferenciação, e analisamos a localização dos RUNX. Por último, investigamos a presença dos componentes da via Notch-Delta na SVZ e sua localização em relação a RUNX3 em célula-tronco neurais. Verificamos que os três RUNX são expressos e apresentam padrão semelhante de localização, sendo encontrados predominantemente no núcleo de células cultivadas por até 7 dias e no citoplasma após 14 dias in vitro (d.i.v.). Avaliamos a localização de RUNX3 em diferentes tipos celulares, identificados por marcadores específicos para células-tronco, neuroblastos e neurônios. Foi observada predominância de marcação nuclear até 7 d.i.v. e citoplasmática com 14 d.i.v., independentemente do tipo celular. Também foi identificada presença de HES1, TLE1 e NOTCH1 na SVZ e coimunolocalização com RUNX3 em células-tronco neurais. A partir dos resultados obtidos até o momento é possível afirmar que os membros da família Runx são expressos por células-tronco neurais e que, possivelmente, essa expressão está relacionada como o processo de diferenciação destas células em neurônios. Além disso, foi observado que os membros da via Notch-Delta (HES1, TLE1 e NOTCH porção citoplasmática) colocalizam com RUNX3.
Runx family members are transcription factors evolutionarily conserved that regulate the expression of genes involved in cell differentiation and cell cycle progression, that may act as transcriptional activators or repressors. The aim of this thesis was to verify whether the expression of the Runx family members (Runx1, 2 and 3) by neural stem cells (NSC) derived from adult mice subventricular zone (SVZ) is related to the differentiation of these cells into neurons. Using NSC cultured as neurospheres we observed Runx family members gene and protein expression by PCR and Western blotting respectively. In order to analyze the subcellular localization of RUNX, neurospheres were induced to adhere to the substrate and were maintained in culture for 1, 7 and 14 days to promote differentiation. Finally, we investigated the presence of Delta-Notch pathway components in the SVZ and their location relative to RUNX3 neural stem cell. By immunocytochemistry we observed that RUNX-1, -2 and -3 exhibited similar pattern of localization, being found predominantly in the nucleus of the cells cultured up to 7 days and in the cytoplasm after 14 days. RUNX3 was localized in different cell types, identified by specific markers for NSC, neuroblasts and neurons. Our results show predominance of nuclear localization after 7 days in vitro (d.i.v) and cytoplasmic expression after 14 d.i.v, regardless of cell type. We also identified the presence of HES1, TLE1 and NOTCH1 in the SVZ which colocalized with RUNX3 in neural stem cells. So far, our results suggest that Runx family members are expressed by NSC in vitro and that the localization and expression are probably related to the stage of differentiation of these cells into neurons. Furthermore, we observed that the members of Notch-Delta (HES1, TLE1 and Notch cytoplasmic portion) have the same subcellular location which RUNX3.
Runx family members are transcription factors evolutionarily conserved that regulate the expression of genes involved in cell differentiation and cell cycle progression, that may act as transcriptional activators or repressors. The aim of this thesis was to verify whether the expression of the Runx family members (Runx1, 2 and 3) by neural stem cells (NSC) derived from adult mice subventricular zone (SVZ) is related to the differentiation of these cells into neurons. Using NSC cultured as neurospheres we observed Runx family members gene and protein expression by PCR and Western blotting respectively. In order to analyze the subcellular localization of RUNX, neurospheres were induced to adhere to the substrate and were maintained in culture for 1, 7 and 14 days to promote differentiation. Finally, we investigated the presence of Delta-Notch pathway components in the SVZ and their location relative to RUNX3 neural stem cell. By immunocytochemistry we observed that RUNX-1, -2 and -3 exhibited similar pattern of localization, being found predominantly in the nucleus of the cells cultured up to 7 days and in the cytoplasm after 14 days. RUNX3 was localized in different cell types, identified by specific markers for NSC, neuroblasts and neurons. Our results show predominance of nuclear localization after 7 days in vitro (d.i.v) and cytoplasmic expression after 14 d.i.v, regardless of cell type. We also identified the presence of HES1, TLE1 and NOTCH1 in the SVZ which colocalized with RUNX3 in neural stem cells. So far, our results suggest that Runx family members are expressed by NSC in vitro and that the localization and expression are probably related to the stage of differentiation of these cells into neurons. Furthermore, we observed that the members of Notch-Delta (HES1, TLE1 and Notch cytoplasmic portion) have the same subcellular location which RUNX3.
Descrição
Citação
MASSINHANI, Fernando Henrique. Indução de diferenciação causa mudança na localização subcelular de runx em células-tronco neurais. 2015. 75 f. Dissertação (Mestrado em Biologia Molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.