Papel dos receptores P2X2 e P2X7 na diferenciação neural in vitro
Nenhuma Miniatura disponível
Arquivos
Data
Autores
Yuahasi, Katia Kioko [UNIFESP]
Orientadores
Ulrich, Alexander Henning [UNIFESP]
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Células-tronco embrionárias e células de carcinomas embrionários são
modelos de diferenciação neural que permitem estudar os mecanismos de
diferenciação, pois mimetizam os eventos críticos durante o início da
embriogênese, adquirindo capacidade de maturação para se tornarem
fisiologicamente funcionais. Esse estudo visa investigar o papel da expressão dos
receptores P2X2 e P2X7 durante a diferenciação neural in vitro em linhagem de
células P19 carcinoma embrionário pela down-regulação da sua expressão,
usando o silenciamento gênico pela técnica do RNA de interferência (RNAi).
Foram desenvolvidas células P19 expressando sequências shRNA interferentes
para os receptores P2X2 e P2X7. Para examinar os efeitos dos silenciamentos
gênicos dos receptores purinérgicos na diferenciação celular analisamos as
expressões gênicas dos marcadores de células indiferenciadas (SSEA-1, antígeno
embriônico estágio específico), progenitores neurais (nestina) e células neurais
(β3-tubulina e GFAP). Além disso, os efeitos de RNAi sobre a proliferação celular
e a atividade de vários receptores ionótropicos e metabótropicos foram avaliados.
O silenciamento gênico dos receptores P2X2 durante a diferenciação neural das
células P19 promoveu aumento da proliferação celular, diminuição significativa da
expressão de proteínas β3-tubulina e elevação da expressão de GFAP, proteína
especificamente expressa por glias, sugerindo que a proliferação promove o
aumento de células gliais. Além disso, houve redução significativa da atividade do
receptor colinérgico, possivelmente relacionada com o bloqueio da diferenciação
de células P19 para neurônios colinérgicos. Portanto, o receptor P2X2 promove a
neurogênese. Diferente do receptor P2X2, a inibição da expressão do receptor
P2X7 durante a diferenciação neural de células P19 levou à redução da
proliferação celular e da expressão de GFAP. Além disso, a expressão de β3-
tubulina aumentou significantemente, sem alterações nos marcadores de células
indiferenciadas (SSEA-1) e de progenitores neurais (nestina). Em células P19
diferenciadas expressando shRNA interferentes para o receptor P2X7, as
respostas de [Ca2+]i induzidas pelo agonista BzATP foram reduzidas, relacionadas
com a inibição da expressão do receptor P2X7. Portanto, esses resultados
sugerem a participação do receptor P2X7 na gliogênese. Esse trabalho confirma a
relevância de sinalização purinérgica na determinação do destino fenotípico da
diferenciação neural, no qual os receptores P2X2 e P2X7 promovem a
neurogênese e a gliogênese, respectivamente. Os shRNA interferindo com a
expressão de receptores P2X2 e P2X7 serão úteis para estudos de mecanismo da
diferenciação neural utilizando outros modelos de células-tronco.
Embryonic stem and carcinoma cells are widely used models for studying the mechanisms of proliferation and differentiation occurring during early embryogenesis, when stem cells acquiring capacity for maturation and physiologically functional patterns of gene expression similar to the cells of the inner layer of the epiblast. The aim of this thesis was to investigate how downregulation of P2X2 and P2X7 receptor expression by gene silencing by interference RNA (RNAi) affects neural differentiation of P19 embryonal carcinoma cells. Thus, we developed P19 embryonal carcinoma cells permanently expressing shRNAs interfering with P2X2 and P2X7 receptor expression. Subsequently, these cells were differentiated into neurons and glial cells by the action of retinoic acid. The P2X2 receptor gene silencing assessed during the neural differentiation of P19 cells promoted an increase in cell proliferation and elevated gene expression of the glial cell marker protein GFAP, suggesting that proliferation is promoting the growth of glial cells. At the same time, we observed a significant decrease in expression of the neuron-specific protein β3-tubulin. Cholinergic receptor activity was largely reduced in differentiated P19 cells expressing P2X2 receptor-specific shRNAs possibly suggesting that maturation into neurons with cholinergic phenotypes was blocked. Differently from the P2X2 receptor, inhibition of P2X7 receptor expression during neural differentiation of P19 cells promoted a decrease in cell proliferation and GFAP expression, suggesting the need for functional P2X7 receptors during gliogenesis. Furthermore, β3-tubulin increased significantly without changes in expression of SSEA-1 and nestin, marker proteins of undifferentiated cells and neural progenitors, respectively. Down-regulation of P2X7 receptor expression contributed to a reduction of Bz-ATP-induced transients of cytosolic calcium concentration ([Ca2+]i). The results obtained in this thesis indicate the importance of purinergic signaling for cell fate determination during neural differentiation, with P2X2 and P2X7 receptors promoting neurogenesis and gliogenesis, respectively. The shRNAs down-regulating P2X2 or P2X7 receptor gene expression, developed during this work, present tools for studying mechanisms of neural differentiation in other stem cell models.
Embryonic stem and carcinoma cells are widely used models for studying the mechanisms of proliferation and differentiation occurring during early embryogenesis, when stem cells acquiring capacity for maturation and physiologically functional patterns of gene expression similar to the cells of the inner layer of the epiblast. The aim of this thesis was to investigate how downregulation of P2X2 and P2X7 receptor expression by gene silencing by interference RNA (RNAi) affects neural differentiation of P19 embryonal carcinoma cells. Thus, we developed P19 embryonal carcinoma cells permanently expressing shRNAs interfering with P2X2 and P2X7 receptor expression. Subsequently, these cells were differentiated into neurons and glial cells by the action of retinoic acid. The P2X2 receptor gene silencing assessed during the neural differentiation of P19 cells promoted an increase in cell proliferation and elevated gene expression of the glial cell marker protein GFAP, suggesting that proliferation is promoting the growth of glial cells. At the same time, we observed a significant decrease in expression of the neuron-specific protein β3-tubulin. Cholinergic receptor activity was largely reduced in differentiated P19 cells expressing P2X2 receptor-specific shRNAs possibly suggesting that maturation into neurons with cholinergic phenotypes was blocked. Differently from the P2X2 receptor, inhibition of P2X7 receptor expression during neural differentiation of P19 cells promoted a decrease in cell proliferation and GFAP expression, suggesting the need for functional P2X7 receptors during gliogenesis. Furthermore, β3-tubulin increased significantly without changes in expression of SSEA-1 and nestin, marker proteins of undifferentiated cells and neural progenitors, respectively. Down-regulation of P2X7 receptor expression contributed to a reduction of Bz-ATP-induced transients of cytosolic calcium concentration ([Ca2+]i). The results obtained in this thesis indicate the importance of purinergic signaling for cell fate determination during neural differentiation, with P2X2 and P2X7 receptors promoting neurogenesis and gliogenesis, respectively. The shRNAs down-regulating P2X2 or P2X7 receptor gene expression, developed during this work, present tools for studying mechanisms of neural differentiation in other stem cell models.
Descrição
Citação
YUAHASI, Katia Kioko. Papel dos receptores purinérgicos P2X2 e P2X7 na diferenciação neural in vitro. 2010. 165 f. Tese (Doutorado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo, 2010,