Estudo da secreção de CXCL12 por astrócitos e células-tronco mesenquimais
Data
2015-06-24
Autores
Reis, Marcella Braga da Costa 
Orientadores
Porcionatto, Marimelia Porcionatto
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O CXCL12 é uma quimiocina presente na maioria dos tecidos de mamíferos. Possui dois receptores conhecidos, o CXCR4 e o CXCR7, porém a maior parte de suas funções está relacionada à ativação do CXCR4. Na neurobiologia, sua importância se deve a sua capacidade de modulação de diferentes etapas da neurogênese como proliferação, migração, diferenciação, extensão neurítica e formação de sinapses. O estudo de suas funções, assim como de sua expressão e secreção são de extrema importância para o entendimento dos processos neuroregenerativos. Neste trabalho construímos vetores para expressão de CXCL12 acoplado a proteína fluorescente em células-tronco mesenquimais (CTM) com o intuito de utilizá-las em lesão cortical isquêmica. Paralelamente, analisamos a secreção de CXCL12 em astrócitos nocaute para conexina 43 (Cx43), proteína previamente relacionada à secreção de CXCL12 por CTM. Observamos inicialmente que os vetores foram produzidos com sucesso e que células HEK293T transfectadas com os vetores construídos expressavam e secretavam a proteína de fusão. Também analisamos a funcionalidade da quimiocina recombinante e, por ensaio de migração em câmara transwell, concluímos que a proteína de fusão secretada possuia atividade quimiotática. Porém, quando aumentamos a expressão de CXCL12 por transdução viral em CTM, detectamos a proteína de fusão apenas dentro da célula, não sendo possível detectar secreção de CXCL12 recombinante por CTM. Também observamos que animais isquêmicos transplantados com CTM contendo a sequência da proteína híbrida não apresentaram aumento de neuroblastos migratórios atraídos para o local do transplante. Adicionalmente, verificamos que a secreção e expressão de CXCL12 por astrócitos imortalizados e provenientes de cultura primária é regulada negativamente pela Cx43 e que essa modulação não interfere na expressão e localização subcelular do Sp1, fator de transcrição responsável pela regulação da expressão do CXCL12.
CXCL12 is a chemokine expressed in many tissues of mammals. This chemokine is able to bind to and activate two receptors, CXCR4 and CXCR7, but its function is mostly related to CXCR4 activation. In neurobiology, the importance of CXCL12 Is related to its ability to modulate different steps of neurogenesis such as proliferation, migration, differentiation, neurite outgrowth and synapses generation. The study of its functions, as well as expression and secretion are extremely important for neuroregeneration research. In this work we promoted the expression of CXCL12 coupled to a fluorescent protein in mesenchymal stem cells (MSC) and transplanted them in ischemic injury of the cortical brain. In addition, we analyzed the secretion of this chemokine in connexin 43 (Cx43) knockout astrocytes, since Cx43 has already been related to CXCL12 secretion by MSC. Initially, we observed that the vectors were successfully produced and that HEK293T cells transfected with the constructs were able to express and secrete the fusion protein. We also analyzed the functionality of the hybrid protein using a transwell chamber in a migration assay and we concluded that the fusion protein had chemotactic activity. However, when we increased CXCL12 expression by viral transduction of MSC, the fusion protein was detected only inside the cells, and no CXCL12 secretion by MSC was detected. We also observed that ischemic mice transplanted with MSC expressing the hybrid protein did not present increase in migratory neuroblasts at the transplantation site. In addition, we found that Cx43 negatively regulates the secretion and expression of CXCL12 by primary and immortalized astrocytes and that this modulation does not interfere with expression and subcellular localization of Sp1 transcription factor, responsible for regulation of CXCL12 expression.
CXCL12 is a chemokine expressed in many tissues of mammals. This chemokine is able to bind to and activate two receptors, CXCR4 and CXCR7, but its function is mostly related to CXCR4 activation. In neurobiology, the importance of CXCL12 Is related to its ability to modulate different steps of neurogenesis such as proliferation, migration, differentiation, neurite outgrowth and synapses generation. The study of its functions, as well as expression and secretion are extremely important for neuroregeneration research. In this work we promoted the expression of CXCL12 coupled to a fluorescent protein in mesenchymal stem cells (MSC) and transplanted them in ischemic injury of the cortical brain. In addition, we analyzed the secretion of this chemokine in connexin 43 (Cx43) knockout astrocytes, since Cx43 has already been related to CXCL12 secretion by MSC. Initially, we observed that the vectors were successfully produced and that HEK293T cells transfected with the constructs were able to express and secrete the fusion protein. We also analyzed the functionality of the hybrid protein using a transwell chamber in a migration assay and we concluded that the fusion protein had chemotactic activity. However, when we increased CXCL12 expression by viral transduction of MSC, the fusion protein was detected only inside the cells, and no CXCL12 secretion by MSC was detected. We also observed that ischemic mice transplanted with MSC expressing the hybrid protein did not present increase in migratory neuroblasts at the transplantation site. In addition, we found that Cx43 negatively regulates the secretion and expression of CXCL12 by primary and immortalized astrocytes and that this modulation does not interfere with expression and subcellular localization of Sp1 transcription factor, responsible for regulation of CXCL12 expression.
Descrição
Citação
REIS, Marcella Braga da Costa. Estudo da secreção de CXCL12 por astrócitos e células-tronco mesenquimais. 2015. 104 f. Tese (Doutorado em Biologia Molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.