Vrednotenje funkcionalnosti transficirane celične linije Jurkat z genskim konstruktom za glukokortikoidni receptor

Horvat, Nika (avtor), Sollner Dolenc, Marija (mentor), Franko, Nina (komentor)

Glukokortikoidi so pomembni regulatorji imunskega odziva, njihovo delovanje pa poteka preko glukokortikoidnega receptorja, ki po vezavi liganda uravnava izražanje številnih genov in vpliva na izločanje citokinov. Celična linija Jurkat zaradi nizke bazalne ravni funkcionalnega glukokortikoidnega receptorja kaže odpornost na glukokortikoide, zato je njena uporabnost za raziskovanje glukokortikoidno posredovanih mehanizmov omejena. Namen magistrske naloge je bil ovrednotiti uspešnost transfekcije in funkcionalnost modificirane celične linije Jurkat GR, v katero je bil z metodo nukleofekcije vnesen genski konstrukt za človeški glukokortikoidni receptor. Primerjali smo izvorno celično linijo Jurkat in modificirano linijo Jurkat GR po izpostavitvi deksametazonu kot agonistu glukokortikoidnega receptorja ter mifepristonu kot njegovemu antagonistu. Celice smo aktivirali z ionomicinom in forbol-12-miristat 13-acetatom. Funkcionalnost receptorja smo ovrednotili na ravni izražanja genov in izločanja citokinov. Z metodo kvantitativne verižne reakcije s polimerazo v realnem času smo analizirali izražanje genov za glukokortikoidni receptor, interlevkin 2, glukokortikoidno inducirano levcinsko zadrgo in kriopirin. Z encimsko-imunsko metodo na trdnem nosilcu smo določili koncentracije izločenih citokinov interlevkina 2 in interlevkina 8. Dobljeni rezultati so pokazali, da modificirana celična linija Jurkat GR izraža funkcionalen glukokortikoidni receptor in se odziva na delovanje deksametazona in mifepristona. V primerjavi z izvorno celično linijo so bile v Jurkat GR celicah izrazitejše spremembe v izražanju glukokortikoidno odzivnih genov, predvsem povečano izražanje glukokortikoidno inducirane levcinske zadrge ter zmanjšano izražanje interlevkina 2. Poleg tega pa je deksametazon v Jurkat GR celicah zmanjšal izločanje interlevkina 2 in interlevkina 8, medtem ko so bili učinki v izvorni celični liniji šibkejši ali odsotni. Dodatek mifepristona je delno ali v celoti zavrl učinke deksametazona, kar dodatno potrjuje funkcionalnost vnesenega receptorja. Na podlagi rezultatov smo ugotovili, da je bila transfekcija celične linije Jurkat uspešna in da modificirana celična linija Jurkat GR predstavlja primeren model za raziskovanje signalnih poti in imunskih učinkov glukokortikoidov na limfocite T. Rezultati prispevajo k boljšemu razumevanju delovanja glukokortikoidnega receptorja ter mehanizmov občutljivosti oziroma odpornosti Jurkat celične linije na glukokortikoide.

• Jezik: Slovenski jezik
• Ključne besede: citokini, deksametazon, glukokortikoidni receptor, Jurkat celice, mifepriston
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FFA - Fakulteta za farmacijo
• Založnik: [N. Horvat]
• Leto izida: 2026
• PID: 20.500.12556/RUL-184332
• UDK: 615:577(043.2)
• COBISS.SI-ID: 283962371
• Datum objave v RUL: 04.07.2026

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Functionality assessment of a Jurkat cell line transfected with a glucocorticoid receptor gene construct

Izvleček

Glucocorticoids are the key regulators of the immune response. They act through the glucocorticoid receptor, which, upon ligand binding, regulates the expression of numerous genes and influences cytokine secretion. The Jurkat cell line exhibits resistance to glucocorticoids due to low basal levels of functional glucocorticoid receptor expression; therefore, its usefulness for studying glucocorticoid-mediated mechanisms is limited. The aim of this master’s thesis was to evaluate the efficiency of transfection and the functionality of a modified Jurkat GR cell line, in which a gene construct encoding the human glucocorticoid receptor was introduced using nucleofection. The parental Jurkat cell line and the modified Jurkat GR cell line were compared following exposure to dexamethasone as a glucocorticoid receptor agonist and mifepristone as its antagonist. Cells were activated using ionomycin and PMA. Receptor functionality was assessed at the level of gene expression and cytokine secretion. Quantitative real-time PCR was used to analyze the expression of genes encoding the glucocorticoid receptor, interleukin-2, glucocorticoid-induced leucine zipper, and NOD-like receptor pyrin domain-containing protein 3. Enzyme-linked immunosorbent assays were used to determine the concentrations of the secreted cytokines interleukin-2 and interleukin-8. The obtained results demonstrated that the modified Jurkat GR cell line expresses a functional glucocorticoid receptor and responds to dexamethasone and mifepristone. Compared to the parental cell line, Jurkat GR cells exhibited more pronounced changes in the expression of glucocorticoid-responsive genes, particularly increased expression of glucocorticoid-induced leucine zipper and decreased expression of interleukin-2. In addition, dexamethasone reduced the secretion of interleukin-2 and interleukin-8 in Jurkat GR cells, whereas these effects were weak or absent in the parental cell line. The addition of mifepristone partially or completely reversed the effects of dexamethasone, further confirming the functionality of the introduced receptor. Based on the results obtained, we concluded that the transfection of the Jurkat cell line was successful and that the modified Jurkat GR cell line represents a suitable model for studying signaling pathways and the immune effects of glucocorticoids. The results contribute to a better understanding of glucocorticoid receptor function and the mechanisms underlying glucocorticoid sensitivity and resistance in the Jurkat cell line.

• Ključne besede: cytokines, dexamethasone, glucocorticoid receptor, Jurkat cells, mifepristone