Adipócitos humanos cultivados e mobilização de cálcio induzida pela angiotensina II e insulina
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2005
Authors
Gaiba, Silvana [UNIFESP]
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Ferreira, Lydia Masako [UNIFESP]
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Dissertação de mestrado
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Abstract
Introdução: O tecido adiposo armazena energia e secreta várias moléculas endócrino/parácrina como a angiotensina II (AII) e expressa receptores como de insulina e de AII (AT1 e AT2). Esses hormônios estão envolvidos em várias patologias tais como a obesidade, hipertensão, diabetes e aterosclerose. Objetivos: Caracterização morfológica
da cultura primária de adipócitos humanos e da resposta ao estímulo dos hormônios AII e
insulina. Métodos: Para caracterização morfológica fez-se a coloração com Oil red O,
gotículas de lipídios, MitoTracker red, mitocôndrias e ERTracker, retículo endoplasmático.
Determinaram-se também as fases do ciclo celular, tamanho e complexidade das
populações das células cultivadas e dispersas através da citometria de fluxo. O cultivo
das células foi realizado de acordo com Hauner et al. (1989). Para as medidas do Ca2+
intracelular foi utilizada a análise por microscopia de fluorescência de alta resolução. As
células adiposas foram caracterizadas por imunocitoquímica utilizando-se anticorpos para
insulina, AT1 da AII, receptor de insulina e lectina conjugados com um marcador
fluorescente. Resultados: Por microscopia confocal verificou-se a presença de várias
gotículas lipídicas, citoesqueleto, mitocôndrias, retículo endoplasmático e membrana
celular preservados, e próprios dos pré adipócitos, Os pré-adipócitos apresentaram
receptores e grânulos de insulina e o receptor AT1. Também se verificou por citometria de
fluxo a amostra de células rescém-dispersas que pôde ser classificada como pré-
adipócito. A avaliação temporal dos níveis de Ca2+ nas células de pré-adipócitos
humanos estimuladas com a AII e insulina induzem respostas transientes no caso da AII
o aumento imediato das [Ca2+]i seguido da diminuição desta resposta e no caso da
insulina apresentou oscilações nas [Ca2+]i na presença deste agonista. Conclusões:
Determinou-se a caracterização morfológica da cultura primária de adipócitos humanos
como pré-adipócitos por:apresentarem morfologia semelhante a fibroblastos e inúmeras
gotículas de lipídios; evidenciado a presença dos receptores de angiotensina II (AT1) e
insulina (RI) por imunofluorescência. As células presentes na amostra do tecido adiposo
apresentaram tamanho e complexidade de pré-adipócitos; Nos pré-adipócitos humanos
cultivados verificou-se o aumento dos níveis intracelulares de Ca2+ que foi transiente para
o estímulo com angiotensina II e oscilatório para a insulina.
Introduction: The adipose tissue stores energy and secretes several endocrine/paracrine molecules as the angiotensin II (AII) and expresses receptors as of insulin and of AII (AT1 and AT2). These hormones are involved in several pathologies such as the obesity, hypertension, diabetes and atherosclerosis. Objectives: Morphologic characterization of the primary culture of human adipocytes and of the answer to the incentive of the AII hormones and insulin. Methods: For morphologic characterization the coloration with Oil red O, lipids droplets, MitoTracker red, mitochondrias and ERTracker, endoplasmic reticulum was made. It also determined the phases of the cellular cycle, size and the populations complexity of the cultivated cells and disperse through the flow cytometry. The cells cultivation was accomplished in agreement with Hauner et al. (1989). For the Ca2+ intracellular measures the analysis by fluorescence microscope of high resolution was used. The adipose cells were characterized by immunocytochemistry using antibodies for insulin, AT1 of AII, insulin receptor and lectina conjugated with a fluorescent marker. Results: For confocal microscope was verified the presence of several lipidics droplets, cytoskeleton, mitochondrias, endoplasmic reticulum and cellular membrane preserved, characteristic on preadipocytes. The preadipocytes presented receptors and insulin granules and AT1 receptor It was also verified by flow cytometry the sample of newly-dispersed cells that could be classified as preadipocyte. The temporary evaluation of the Ca2+ levels in the human adipocytes cells which were stimulated with AII and insulin showed that AII induced transient answers, by the immediate increase of the [Ca2+]i followed by the decrease of this response. The insulin presented oscillations in the [Ca2+]i during the presence of this agonist. Conclusions: For confocal microscopy was determined the morphologic characterization of the primary culture of human adipocytes as preadipocytes and evidenced the presence of the receivers of angiotensina II (AT1) and insulin (RI) for imunofluorescência. The present cells in the sample of the fatty fabric presented size and preadipocytes complexity; In the cultivated human preadipocytes the increase of the levels intracelulares of Ca2+ was verified that was transient for the incentive with angiotensina II and oscillatory for the insulin.
Introduction: The adipose tissue stores energy and secretes several endocrine/paracrine molecules as the angiotensin II (AII) and expresses receptors as of insulin and of AII (AT1 and AT2). These hormones are involved in several pathologies such as the obesity, hypertension, diabetes and atherosclerosis. Objectives: Morphologic characterization of the primary culture of human adipocytes and of the answer to the incentive of the AII hormones and insulin. Methods: For morphologic characterization the coloration with Oil red O, lipids droplets, MitoTracker red, mitochondrias and ERTracker, endoplasmic reticulum was made. It also determined the phases of the cellular cycle, size and the populations complexity of the cultivated cells and disperse through the flow cytometry. The cells cultivation was accomplished in agreement with Hauner et al. (1989). For the Ca2+ intracellular measures the analysis by fluorescence microscope of high resolution was used. The adipose cells were characterized by immunocytochemistry using antibodies for insulin, AT1 of AII, insulin receptor and lectina conjugated with a fluorescent marker. Results: For confocal microscope was verified the presence of several lipidics droplets, cytoskeleton, mitochondrias, endoplasmic reticulum and cellular membrane preserved, characteristic on preadipocytes. The preadipocytes presented receptors and insulin granules and AT1 receptor It was also verified by flow cytometry the sample of newly-dispersed cells that could be classified as preadipocyte. The temporary evaluation of the Ca2+ levels in the human adipocytes cells which were stimulated with AII and insulin showed that AII induced transient answers, by the immediate increase of the [Ca2+]i followed by the decrease of this response. The insulin presented oscillations in the [Ca2+]i during the presence of this agonist. Conclusions: For confocal microscopy was determined the morphologic characterization of the primary culture of human adipocytes as preadipocytes and evidenced the presence of the receivers of angiotensina II (AT1) and insulin (RI) for imunofluorescência. The present cells in the sample of the fatty fabric presented size and preadipocytes complexity; In the cultivated human preadipocytes the increase of the levels intracelulares of Ca2+ was verified that was transient for the incentive with angiotensina II and oscillatory for the insulin.
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GAIBA, Silvana. Adipócitos humanos cultivados e mobilização de cálcio induzida pela angiotensina II e insulina. 2005. 112 f. Dissertação (Mestrado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2005.