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Authors
Advisor(s)
Abstract(s)
No presente relatório avaliou-se a pertinência da implementação das células F-11, um hibridoma derivado de neuroblastoma de ratinho e neurónios do gânglio da raiz dorsal de embriões de rato, como um modelo funcional, alternativo a neurónios de DRG, para a investigação e desenvolvimento de novas moléculas terapêuticas para o tratamento da dor. Recorrendo a uma abordagem técnica multidisciplinar, que incluiu cultura de células, técnicas de eletrofisiologia (whole-cell voltage-clamp e current-clamp) e técnicas de biologia molecular (western-blotting) foi possível estudar a influência do processo de diferenciação das células F-11 sobre o perfil bioquímico e eletrofisiológico das correntes iónicas dependentes de voltagem, responsáveis pela modulação da fisiopatologia da dor. O meio de crescimento F-12, suplementado com 10%FBS, e os meios de diferenciação DMEM, suplementado com 1% FBS, e DMEM, suplementado com 2%FBS e 2μM RA, permitiram registos de eletrofisiologia com os menores valores médios de holding current (Ih (pA)). A comparação dos resultados obtidos entre as células destes meios revelou um aumento da amplitude de correntes lentas de potássio e correntes de sódio totais em células F-11 diferenciadas. Esta observação explica-se pela variação da expressão proteica de alguns canais de potássio e sódio durante o processo de diferenciação. Por outro lado, a ausência de correntes de cálcio nas células F-11 sustenta a reduzida atividade neuronal, traduzida num registo quase nulo de potenciais de ação, tanto nas células não diferenciadas como nas diferenciadas. Os resultados sugerem ainda que as células F11 e os neurónios de DRG partilham o mesmo tipo de correntes lentas de potássio e o mesmo tipo de correntes de sódio sensíveis ao fármaco Tetrodotoxina. Os resultados demonstram que a linha celular F-11, mantida e diferenciada nos meios analisados, pode ser usada como uma plataforma de investigação no âmbito da descoberta de novas moléculas terapêuticas para o tratamento da dor, sobretudo no que respeita ao envolvimento de canais de sódio e de potássio.
ABSTRACT - In this report, the relevance of implementing F-11 cells, a hybridoma derived from mice’s neuroblastoma and embryonic rat dorsal root ganglion neurons, as a functional model, in alternative to dorsal root ganglion neurons, for research and development of new therapeutic molecules for the treatment of pain. Using a multidisciplinary technical approach, which included cell culture, electrophysiology techniques (whole cell voltage clamp and current clamp), and molecular biology techniques (western blotting), it was possible to study the influence of the differentiation process of F-11 cells on the electrophysiological and biochemical profile of voltage-gated ion currents, responsible for modulating the pathophysiology of pain. The growth medium F-12, supplemented with 10%FBS and the differentiation mediums DMEM, supplemented with 1% FBS and DMEM supplemented with 2% FBS and 2µM RA, allowed electrophysiology recordings with the lowest average holding current (Ih (pA)) values. Comparison between the results obtained from cells in these mediums revealed an increase in the amplitude of slow potassium currents and total sodium currents in differentiated F-11 cells. This observation can be explained by the variation in protein expression of some potassium and sodium channels during the differentiation process. On the other hand, the absence of calcium currents in F-11 cells underpins the reduced neuronal activity, reflected in the almost null recording of action potentials in both differentiated and undifferentiated cells. The results also suggest that F-11 cells and dorsal root ganglion neurons share the same type of slow potassium currents and the same type of sodium currents sensitive to the drug Tetrodotoxin. The results show that the F-11 cell line, kept and differentiated in the analyzed mediums, can be used as a research platform for the discovery of new therapeutic molecules in pain treatment, especially regarding the involvement of sodium and potassium channels.
ABSTRACT - In this report, the relevance of implementing F-11 cells, a hybridoma derived from mice’s neuroblastoma and embryonic rat dorsal root ganglion neurons, as a functional model, in alternative to dorsal root ganglion neurons, for research and development of new therapeutic molecules for the treatment of pain. Using a multidisciplinary technical approach, which included cell culture, electrophysiology techniques (whole cell voltage clamp and current clamp), and molecular biology techniques (western blotting), it was possible to study the influence of the differentiation process of F-11 cells on the electrophysiological and biochemical profile of voltage-gated ion currents, responsible for modulating the pathophysiology of pain. The growth medium F-12, supplemented with 10%FBS and the differentiation mediums DMEM, supplemented with 1% FBS and DMEM supplemented with 2% FBS and 2µM RA, allowed electrophysiology recordings with the lowest average holding current (Ih (pA)) values. Comparison between the results obtained from cells in these mediums revealed an increase in the amplitude of slow potassium currents and total sodium currents in differentiated F-11 cells. This observation can be explained by the variation in protein expression of some potassium and sodium channels during the differentiation process. On the other hand, the absence of calcium currents in F-11 cells underpins the reduced neuronal activity, reflected in the almost null recording of action potentials in both differentiated and undifferentiated cells. The results also suggest that F-11 cells and dorsal root ganglion neurons share the same type of slow potassium currents and the same type of sodium currents sensitive to the drug Tetrodotoxin. The results show that the F-11 cell line, kept and differentiated in the analyzed mediums, can be used as a research platform for the discovery of new therapeutic molecules in pain treatment, especially regarding the involvement of sodium and potassium channels.
Description
Mestrado em Tecnologias Clínico-Laboratoriais
Keywords
Células F-11 Canais iónicos Voltage-clamp Current-clamp Neurónios DRG Excitabilidade F-11 cells Ionic channels DRG neurons Excitability
Pedagogical Context
Citation
Bernardo AF. Estágio em neurofisiologia e microbiologia: implementação da linha celular imortalizada F-11 em estudos da dor [dissertation]. Lisboa: Escola Superior de Tecnologia da Saúde de Lisboa/Instituto Politécnico de Lisboa; 2023.
Publisher
Instituto Politécnico de Lisboa, Escola Superior de Tecnologia da Saúde de Lisboa