Existem algumas limitações inerentes ao estudos de envelhecimento realizados in vitro. Na verdade, estes estudos são bastante morosos e não podem ser repetidos, a não ser que a cada subcultura sejam armazenadas em azoto líquido alíquotas correspondentes às passagens celulares testadas. Ainda assim, terá sempre de se ter em conta o stress provocado pelos processos de congelamento, descongelamento e tripsinização associada.
49
Conclusões
Através do estudo da resposta de culturas de fibroblastos NHDF ao 17β-estradiol pode concluir-se o seguinte:
A reprodutibilidade entre a resposta de quatro passagens celulares contíguas de NHDF apenas foi verificada, quando as culturas foram incubadas em meio suplementado com FBS e expostas ao E2 por 48 horas.
A relação entre os valores de absorvência de MTT e número de células contabilizado só foi assegurada quando os fibroblastos foram incubados em meio suplementado com FBS nos períodos de 48 e 96 horas.
A resposta dos fibroblastos ao E2 somente se mostrou significativa após 96 horas de incubação em meio suplementado com FBS.
50
Protocolo ideal
As condições experimentais ideais para a utilização dos fibroblastos NHDF em cultura, como modelo de estudo da resposta a estrogénios devem respeitar os seguintes parâmetros:
De forma a garantir a reprodutibilidade dos ensaios, assim como a correspondência entre valores de absorvência de MTT e número de células, as culturas devem ser sempre incubadas em meio suplementado com FBS.
Uma vez que para além da reprodutibilidade, a sensibilidade aos estrogénios também é um factor fundamental, deve ser estabelecido um equilíbrio entre a reprodutibilidade e a sensibilidade. Para tal, se o tempo de incubação escolhido for de 96 horas a falta de reprodutibilidade deve ser compensada com a realização de um maior número de estudos. Por outro lado, se os ensaios forem iniciados com um menor número de células pode conseguir-se um aumento de sensibilidade às 48 horas, garantindo-se assim a reprodutibilidade.
De forma a reduzir a variabilidade entre a resposta de diferentes passagens o ideal seria que todas as experiências de um mesmo ensaio fossem realizadas na mesma passagem celular.
51
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57
Anexo
Tabela 6 - Valores de t aos Níveis de significância de 0,05 e 0,01.
gl 0,05 0,01 1 12,706 63,657 2 4,303 9,925 3 3,182 5,841 4 2,776 4,604 5 2,571 4,032 6 2,447 3,707 7 2,365 3,499 8 2,306 3,355 9 2,262 3,250 10 2,228 3,169 11 2,201 3,106 12 2,179 3,055 13 2,160 3,012 14 2,145 2,977 15 2,131 2,947 16 2,120 2,921 17 2,110 2,898 18 2,101 2,878 19 2,093 2,861 20 2,086 2,845 120 1,980 2,617 ∞ 1,960 2,576