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Master's Dissertation
DOI
10.11606/D.10.2008.tde-19112008-171339
Document
Author
Full name
Elmer Alexander Genoy-Puerto
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2008
Supervisor
Committee
Matushima, Eliana Reiko (President)
Catão-Dias, José Luiz
Rocha, Vanessa de Moura Sá
Title in Portuguese
Citometria de fluxo de leucócitos sangüíneos de Phrynops geoffroanus (Schweigger, 1812) provenientes de ambientes poluídos: metodologia de isolamento e estimulação
Keywords in Portuguese
Phrynops geoffroanus
Citometria de fluxo
Função celular
Leucócitos sangüíneos
Poluição
Abstract in Portuguese
O quelônio Phrynops geoffroanus aparece freqüentemente associado a cursos d'água poluídos, sem que, no entanto, sejam conhecidos aspectos que descrevam sua atividade imune celular frente situações adversas (efeitos antrópicos). A citometria de fluxo permite mensurar características estruturais e imunológicas de células em suspensão submetidas a um fluxo contínuo, sendo capaz de analisar inúmeros tipos celulares. Neste trabalho foi desenvolvida metodologia para estudar e avaliar a função celular de leucócitos sangüíneos do Phrynops geoffroanus através da fagocitose e burst oxidativo espontâneo e induzido utilizando a citometria de fluxo. Para definir a metodologia foram utilizadas 86 amostras sangüíneas de Phrynops geoffroanus encontrados em ambientes antrópicos do rio Piracicaba, ribeirão Piracicamirim e Fundação Parque Zoológico de São Paulo como grupo de comparação. Estes resultados foram complementados pelo perfil hematológico. O processamento incluiu transporte e conservação das amostras em RPMI 1640 Gibco® para posterior utilização de Ficoll-PaqueTM PLUS como agente separador entre leucócitos e hemácias, utilizando-se centrifugações refrigeradas (18°C) com acelerações e desacelerações graduais. Amostras com porcentagens de viabilidade inferiores a 90 % não foram utilizadas na realização dos estímulos: Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate para induzir fagocitose e miristato acetato de phorbol (PMA) e Saccharomyces cerevisiae (Zymosan) na indução do burst oxidativo. Condições ambientais, espécie específica e da metodologia da citometria de fluxo determinaram uma melhor resposta para realização da fagocitose em comparação com o burst oxidativo dos leucócitos aqui avaliados.
Title in English
Flow cytometry of blood leucocytes of Phrynops geoffroanus (Schweigger, 1812) from polluted environments: isolation and stimulation methodology
Keywords in English
Phrynops geoffroanus
Blood leucocytes
Cellular function
Flow cytometry
Pollution
Abstract in English
The freshwater turtle Phrynops geoffroanus is frequently associated with polluted waters; however, the aspects that describe the cellular immune activity against adverse situations (anthropogenic effects) are still unknown. Flow cytometry allows us to measure and analyze the characteristics of the suspended cells when submitted to a continuous flow; it is capable of analyzing a number of different types of cells. In this study, it designed a methodology in order to study and evaluate the cellular function of blood leucocytes, through phagocytosis and spontaneous or induced oxidative bursts, using flow cytometry. This methodology, was designed with 86 blood samples, collected from Phrynops geoffroanus in anthropogenic environments of the river Piracicaba and its tributary Piracicamirim and the Sao Paulo Zoological Park Foundation, as a comparison group, all in Sao Paulo State, Brazil. These results were complemented with the blood profile. The samples were stored in RPMI 1640 Gibco®, medium for transportation. Initially, it was used Ficoll-PaqueTM PLUS, as a separator of leucocytes and red blood cells. Later the samples were centrifuged at 18°C, with gradual break and speed. Samples with viability percentage under 90% were not used. The agents used for stimulation were Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate to induce phagocytosis and phorbol miristate-acetate (PMA) and Saccharomyces cerevisiae (Zymosan) to induce oxidative burst. Environmental, species-specific and cytometric methodology conditions determined a better response for the occurrence of phagocytosis in comparison with oxidative burst of the leucocytes analyzed.
 
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Publishing Date
2009-04-15
 
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