magistrsko delo
Ana Špilak (Author), Peter Maček (Reviewer), Marjetka Podobnik (Mentor), Marjetka Podobnik (Thesis defence commission member), Apolonija Bedina Zavec (Thesis defence commission member), Peter Maček (Thesis defence commission member), Tom Turk (Thesis defence commission member), Apolonija Bedina Zavec (Co-mentor)

Abstract

Zunajcelični vezikli (ZV) so pomembni faktorji pri vrednotenju celičnega stanja. V magistrski nalogi smo želeli ugotoviti ali se števili večjih (vZV) in manjših zunajceličnih veziklov (mZV) spreminjata po izpostavitvi celic porotvornim proteinom (PFP) listeriolizinu O (LLO) in njegovima mutantoma LLOY406A in LLOA318C-L334C. Dokazano je, da obdelava celic z nekaterimi PFP povzroča povečano izločanje ZV, ni pa še znano, kako se celice odzovejo na LLO. Dosedanje študije so pokazale, da je LLOY406A pri fiziološkem pH neaktiven, pri znižanju pH pa se njegova aktivnost poveča. LLOA318C-L334C je v oksidiranem stanju zakljenjen v strukturo, ki onemogoča nastanek pore, v reduciranem stanju pa lahko tvori pore. Odziv celic na LLO in mutanta smo ovrednotili z določanjem viabilnosti celic in s spremljanjem količine ZV, ki so jih celice sprostile po izpostavitvi toksinom. Uporabili smo sesalske celične linije K-562, Jurkat in Caco-2. Viabilnost in vezikulacijo celic smo ugotavljali s pretočnim citometrom, dinamičnim sipanjem svetlobe, analizo sledenja nanodelcem in s konfokalnim mikroskopom. Ugotovili smo, da je preživetje celic najmanjše po učinkovanju z LLO pri pH 6, sledita mu LLO pri pH 7,4 in LLOY406A pri pH 6. LLOA318C-L334C ox prav tako vpliva na viabilnost celic, vendar v manjši meri, LLOY406A pri pH 7,4 pa ni litičen oziroma je šibko citolitičen. Po obdelavi celic z litičnimi koncentracijami LLO (pH 7,4 in pH 6) in LLOY406A (pH 6) celice močno vezikulirajo in izločajo več vZV in mZV glede na kontrolo. Pri koncentracijah, ki so 100-krat manjše od litične, pa celice v večini primerov izločijo manj vZV in mZV kot kontrolne. Odziv celic na LLOA318C-L334C ox je po količini izločenih ZV podoben kot pri kontroli. Dolgoročno bodo rezultati raziskave lahko doprinesli k uporabi PFP v farmacevtski industriji in v medicinske namene.

Keywords

zunajcelični vezikli;listeriolizin O;sesalske celične linije;celični odziv;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [A. Špilak]
UDC: 577(043.2)
COBISS: 5046351 Link will open in a new window
Views: 982
Downloads: 293
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Other data

Secondary language: English
Secondary title: Viability and vesiculation of mammalian cells after exposure with protein listeriolysin O
Secondary abstract: Extracellular vesicles (EV) are important factors in characterization of cell condition. In this master thesis we wanted to discover whether the number of large extracellular vesicles (lEV) and/or small extracellular vesicles (sZV) changes after exposing cells to pore forming proteins (PFP) listeriolysin O (LLO) and its two mutants: LLOY406A and LLOA318C-L334C. There was already shown that treating cells with some PFP can lead to higher excretion of EV, but not jet for LLO. Activity of LLO depends on the protein concentration, membrane composition, pH and temperature. In this research project we focused on the effect of wild type LLO and its two mutants LLOY406A and LLOA318C-L334C on cell lines K-562, Jurkat and Caco-2. Previous studies have shown that LLOY406A works similarly as LLO at acidic pH, but at physiological pH it is inactive. LLOA318C-L334C is, when oxidized, locked in such a conformation that pore formation cannot be completed, while in the reduced state it acts similarly as LLO. We characterized response of the cells treated with selected proteins through observing viability and vesiculation using flow cytometry, dynamic light scattering, nanoparticle tracking analysis, and light and confocal microscopy. We found that the cell survival is the lowest after treating cells with LLO at pH 6, followed by LLO at pH 7,4 and LLOY406A at pH 6. LLOA318C-L334C ox can also effect the viability of the cells, but in a smaller scale, and LLOY406A is not litic for the cells at pH 7,4. After treating cells with lytic concentrations of LLO and LLOY406A cells heavily vesiculated and produced more lEV and sEV in comparison to control. At concentrations that were 100-fold lower than lytic ones, cells usually produced less lEV and sEV than control. Cell response to oxidized LLOA318CL334C regarding vesiculation is similar to controls. In future, these findings could helpful at determining cell response to PFP for potential use in the farmaceutical industry.
Secondary keywords: extracellular vesicles;listeriolysin O;mammalian cell culture;cell response;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. Ljubljana, Biotehniška fak.
Pages: X f., 70, [1] str.
ID: 11061056