hrvatski

Dokazivanje antitumorskog učinka neutrofila

Oxidative stress (state of excess in production of reactive oxygen species (ROS)) is generaly considered as an important part of different kind of diseases (neurodegenerative diseases, malignant tumors, cardiovascular diseases, diabetes mellitus, traumatic injuries etc.). However, inflammatory processes (organism reaction on potentially harmful factors) include oxidative stress as physiological event. It is supposed that the basic difference between physiological and pathological oidative stress is in control of lipid peroxidation and its final toxic products, among which is reactive aldehyde 4-hydroxi-2-nonenal (HNE), physiologically produced “ second toxic messenger of free radicals” . However formation of HNE and its role in inflammatory processes is not yet understood. The goal of this investigation was to determine ROS (superoxide anion, reactive (singlet) oxigen) in neutrophils, in vivo, with nitroblue tetrasolium test and chemiluminiscence method. We also wanted to determine subcellular morphological distribution of HNE-hystidine conjugates, in neutrophils from full blood, with immunogold method. For immunocytochemical and immunoelectromicroscopical analysis of HNE formation in inflammatory cells monoclonal antibodies against HNE-hystidine conjugates were used. Neutrophils from full blood and neutrophils from rat’ s papula (rat was earlier supcutaneously injected with sephadex G-200) were analysed. More than 93% of cells in inflammatory tissue area were neutrophils. Nitroblue tetrasolim test showed that, from all activated neutrophils, 97% produced superoxide anion (O2• -). Chemiluminiscence method showed that production of reactive oxigen species (1O2) was growing by rising the number of activated neutrophils versus melanoma B16 cells. However the production of reactive singlet oxigen in nonactivated neutrophils was also rising when melanoma B16 cells were aded. Immunicytochemistry PAP metod showed that there were no HNE-histidine conjugates in nonactivated neutrophils, activated neutrophils showed great HNE-positivity in membrane area. Electron microscopy analises showed that formations of HNE-hystidine conjugates were mostly seen near phagosomes and macrophage’ s granula. This immunoelectromicroscopy of HNE-hystidine conjugates could be used as analitical method to study pathological and physiological roles of HNE, as well as oxidative stress.

neutrofili; stanice melanoma B16; reaktivni kisikovi spojevi; oksidativni stres; fagocitoza

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