Investigação da influência do zinco na participação do cininogênio humano de alta massa molecular em eventos celulares
Data
2015-10-28
Autores
Souza, Daianne Santos Paranhos de 
Orientadores
Motta, Guacyara da
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
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Resumo
Objetivo: o objetivo geral do presente trabalho é estudar a influência do íon zinco na interação celular, mediada por proteoglicanos, do cininogênio de alta massa molecular íntegro (HK) ou livre de bradicinina (HKa). Métodos: identificar a atividade enzimática de células de diferentes linhagens epiteliais de tumor de ovário de hamster chinês sobre o cininogênio de alto peso molecular, de forma intracelular (através do lisado) ou extracelular (na superfície de células em cultura) através de eletroforese em gel de poliacrilamida na presença de dodecil sulfato de sódio e imunodetecção; estudar a influência do íon zinco na endocitose de cininogênio de alto peso molecular (através de microscopia confocal) e os efeitos do íon em eventos celulares como morte celular, apoptose e viabilidade em células em cultura, através de ensaios utilizando o corante Trypan Blue, anexina V e o reagente MTT, respectivamente. Resultados: ambas as linhagens liberam bradicinina a partir do cininogênio de alto peso molecular na superfície celular pela ação de serino- e cisteinoproteases. O cininogênio de alto peso molecular íntegro e cininogênio de alto peso molecular livre de cinina são internalizados pelas células epiteliais de tumor de ovário de hamster chinês selvagens, localizando-se em vesículas endocíticas ácidas, indicando a importância do proteoglicano de heparam sulfato como mediador. A hidrólise do cininogênio de alto peso molecular íntegro foi estudada através de frações de membrana e lisados, preparados a partir dessas linhagens celulares, onde observamos que: (1) enzimas de membrana e de lisado das duas linhagens hidrolisam o HK em locais diferentes; (2) a atividade cininogenásica é influenciada pelo pH na fração de lisado de ambas as linhagens; (3) atividade cininogenásica difere entre estas duas linhagens celulares sugerindo a influência de glicosaminoglicanos no processamento do cininogênio de alto peso molecular; (4) tanto a molécula de cininogênio de alto peso molecular quanto o íon zinco influenciam eventos fisiológicos como morte e viabilidade celular. Conclusão: nossos resultados mostram que o íon zinco na presença de PGs (CHO-K1) promove a endocitose do HK na sua forma íntegra com fraca hidrólise e este HK endocitado reduz a viabilidade celular provocando necrose, sendo que o zinco pode amenizar esse efeito.
Objective: the general objective of this work is studying the influence of zinc in the cell interaction mediated by proteoglycans, of whole molecule of high molecular weight kininogen (HK) or kinin-free (HKa). Methods: identifying the enzymatic activity of epithelial cells from different strains of Chinese hamster ovarian tumor on high molecular weight kininogen, inside the cell (by lysate) or outside the cell (on the surface of cells in culture), using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and immunodetection; studying the influence of the zinc ion in the endocytosis of high molecular weight kininogen (via confocal microscopy), and the ion effects on cellular events such as cell death, apoptosis and viability of cells in culture by assays using Trypan Blue, Annexin V and MTT reagent, respectively. Results: both strains release bradykinin from high molecular weight kininogen at the cell surface by the action of serino- and cisteinoproteases. The whole high molecular weight kininogen and kinin-free high molecular weight kininogen are internalized by the epithelial cells of wild Chinese hamster ovarian tumor being located in acidic endocytic vesicles, indicating the importance of heparan sulfate proteoglycan as a mediator of this process. Hydrolysis of high molecular weight kininogen was studied through membrane and lysates fractions prepared from these cell lines, where we observed that: (1) enzymes in membrane and in lysate of the two strains hydrolyze HK in different locations; (2) kininogenase activity is influenced by pH in the lysate fraction of both strains cells; (3) kininogenase activity is different between the two cell lines, which had suggested the influence of glycosaminoglycans in the processing of high molecular weight kininogen; (4) both kininogen molecule of high molecular weight and the zinc ion influences physiological events such as cell death and viability. Conclusion: our results show that zinc ion in the presence of PGs (CHO-K1) promotes the endocytosis of whole HK molecule with poor hydrolysis and the internalized HK reduces cell viability causing necrosis, and zinc can mitigate this effect.
Objective: the general objective of this work is studying the influence of zinc in the cell interaction mediated by proteoglycans, of whole molecule of high molecular weight kininogen (HK) or kinin-free (HKa). Methods: identifying the enzymatic activity of epithelial cells from different strains of Chinese hamster ovarian tumor on high molecular weight kininogen, inside the cell (by lysate) or outside the cell (on the surface of cells in culture), using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and immunodetection; studying the influence of the zinc ion in the endocytosis of high molecular weight kininogen (via confocal microscopy), and the ion effects on cellular events such as cell death, apoptosis and viability of cells in culture by assays using Trypan Blue, Annexin V and MTT reagent, respectively. Results: both strains release bradykinin from high molecular weight kininogen at the cell surface by the action of serino- and cisteinoproteases. The whole high molecular weight kininogen and kinin-free high molecular weight kininogen are internalized by the epithelial cells of wild Chinese hamster ovarian tumor being located in acidic endocytic vesicles, indicating the importance of heparan sulfate proteoglycan as a mediator of this process. Hydrolysis of high molecular weight kininogen was studied through membrane and lysates fractions prepared from these cell lines, where we observed that: (1) enzymes in membrane and in lysate of the two strains hydrolyze HK in different locations; (2) kininogenase activity is influenced by pH in the lysate fraction of both strains cells; (3) kininogenase activity is different between the two cell lines, which had suggested the influence of glycosaminoglycans in the processing of high molecular weight kininogen; (4) both kininogen molecule of high molecular weight and the zinc ion influences physiological events such as cell death and viability. Conclusion: our results show that zinc ion in the presence of PGs (CHO-K1) promotes the endocytosis of whole HK molecule with poor hydrolysis and the internalized HK reduces cell viability causing necrosis, and zinc can mitigate this effect.
Descrição
Citação
SOUZA, Daianne Santos Paranhos de. Investigação da influência do zinco na participação do cininogênio humano de alta massa molecular em eventos celulares. 2015. 108 f. Dissertação (Mestrado em Biologia Molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.