Acta Univ. Agric. Silvic. Mendelianae Brun. 2009, 57(1), 97-104 | DOI: 10.11118/actaun200957010097

VYUŽITÍ METODY WESTERN BLOT PRO DETEKCI PROTEINŮ SOUVISEJÍCÍCH S APOPTÓZOU NEUTROFILŮ

Petr Sláma1,2
1 Ústav morfologie, fyziologie a genetiky zvířat, Mendelova zemědělská a lesnická univerzita v Brně, Zemědělská 1, 613 00 Brno, Česká republika
2 Výzkumný ústav veterinárního lékařství, v.v.i., Oddělení imunologie, Hudcova 70, 621 00 Brno, Česká republika

Práce byla zaměřena na zjištění vhodnosti použití metody Western Blot pro detekci proteinů souvisejících s apoptózou neutrofilů, jako doplňkové metody pro detekci apoptózy neutrofilů. Neutrofily byly izolovány z humánní krve pěti zdravých dospělých dárců. Dále byly inkubovány s G-CSF (50 ng / ml), GM-CSF (5 ng / ml), ATP (250 μM) a FMLP (10-7 M). Inkubace probíhala 4, 8 a 20 hodin při teplotě 37 °C. Byla sledována exprese proteinů Mcl-1, XIAP a gelsolin. V experimentech docházelo k degradaci proteinu Mcl-1 v závislosti na čase jak u kontrolních vzorků, tak u pokusných vzorků, dále pak v závislosti na použitých činitelích. G-CSF a GM-CSF oddalují apoptózu neutrofilů, což dobře dokládá denzita bandů proteinu Mcl-1. Detekce exprese proteinu Mcl-1 se jeví jako vhodný marker apoptózy neutrofilů. K degradaci XIAP docházelo také v závislosti na čase a použitých činitelích. Zároveň s degradací tohoto proteinu se objevil jeho fragment, který vytvářel samostatný band, který zřetelně ukazuje nastupující apoptózu neutrofilů především po 20 hodinách inkubace. Čím je tento band výraznější, tím intenzivnější je apoptóza neutrofilů. Také při detekci gelsolinu docházelo ke snižování denzity bandů v závislosti na čase a použitých činitelích a vytvářely se další bandy (fragmenty), které jsou markerem apoptózy.
Jak je z výsledků zřejmé, metoda Western Blot je velmi vhodná pro detekci proteinů souvisejících s apoptózou neutrofilů (Mcl-1, XIAP, gelsolin), tzn. že je vhodná i pro posouzení výskytu apoptózy neutrofilů. Stává se tak další významnou metodou studia apoptózy neutrofilů.

Western Blot, neutrofil, apoptóza, cytokiny, Mcl-1, XIAP, gelsolin

Application of Western Blot for detection of neutrophil apoptosis-related proteins

The aim of this study was to evaluate suitability of using Western Blot for detection of neutrophil apoptosis and neutrophil apoptosis-related proteins, respectively. Neutrophils were isolated from blood of healthy adult donors and incubated with G-CSF (granulocyte colony stimulating factor), GM-CSF (granulocyte-macrophage colony stimulating factor), ATP (adenosine triphosphate) and FMLP (N-formyl-methionyl-leucyl-phenylalanine). The neutrophils were incubated 4, 8 and 20 hours at 37 °C. In this assay, an expression of Mcl-1 (myeloid cell leukemia 1), XIAP (X-linked inhibitor of apoptosis) and gelsolin was analysed by Western Blot method. The results showed that Western Blot is a suitable method for detection of neutrophil apoptosis-related proteins and detection of neutrophil apoptosis, respectively.

Keywords: Western Blot, neutrophil, apoptosis, cytokines, Mcl-1, XIAP, gelsolin

Received: December 19, 2008; Published: October 14, 2014  Show citation

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Sláma, P. (2009). Application of Western Blot for detection of neutrophil apoptosis-related proteins. Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis, 57(1), 97-104. doi: 10.11118/actaun200957010097
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