20.500.12556/RUL-173749 Zaznavanje genotoksičnega delovanja ksenobiotikov z naprednim jetrnim in vitro 3D celičnim modelom Detection of genotoxic effects of xenobiotics using an advanced in vitro 3D hepatic cell model Ksenobiotiki predstavljajo pomembno tveganje za zdravje ljudi zaradi svoje sposobnosti povzročanja poškodb DNK, ki lahko vodijo v rakava obolenja, kronične bolezni in druge patološke spremembe. Konvencionalni 2D celični modeli in in vivo živalski poskusi imajo številne omejitve pri ponazarjanju človeške presnove, zato in vitro 3D modeli predstavljajo napredno alternativo pri ocenjevanju genotoksičnosti. V okviru magistrskega dela smo optimizirali in validirali dinamičen in vitro 3D model sferoidov iz celične linije HepG2, ki smo ga uporabili za preučevanje genotoksičnih učinkov policikličnih aromatskih ogljikovodikov (PAH). V prvem raziskovalnem sklopu smo spremljali rast, diferenciacijo in presnovno aktivnost sferoidov tekom njihovega gojenja. Rezultati so pokazali celovito stabilnost sferoidov tekom njihovega gojenja, postopno zorenje in največjo presnovno aktivnost med 17. in 22. dnem starosti sferoidov, potrjeno z izražanjem jetrnih markerjev (ALB, CK18, HNF4α) ter induciranih presnovnih encimov (CYP1A1, CYP3A4, NAT2, UGT1A1). Na podlagi teh rezultatov smo kot optimalno izbrali 21 dni stare sferoide. V drugem raziskovalnem sklopu smo sferoide izpostavili PAH-om benzo(g)perilen (BGP), benzo(b)fluoranten (BBF) in njuno binarno mešanico ter benzo(a)piren (BaP) za 24 in 96 ur. Z uporabo metode pretočne citometrije smo pokazali, da BGP poveča P21- in γH2AX-pozitivnih celic, medtem ko BBF in BaP dodatno povečata procent GADD45α-pozitivnih celic. Mešanica ni izkazala sinergističnih učinkov. Višje koncentracije so povzročile izrazitejše odzive, medtem ko so rezultati dolgotrajne izpostavitve nakazovali bodisi aktivacijo popravljalnih mehanizmov bodisi akumulacijo poškodb, odvisno od vrste PAH-a. Na podlagi izraženih markerjev smo ocenili genotoksični potencial v zaporedju: BGP < BBF < BaP. Xenobiotics represent a significant health risk due to their ability to induce DNA damage, which can lead to cancer, chronic disease and other pathologies. Conventional 2D cell models as well as in vivo animal testing have several limitations in mimicking the human metabolism. Thus in vitro 3D models represent an innovative alternative for genotoxicity assessment. In this master’s thesis we optimised and validated a dynamic in vitro 3D spheroid model constructed from the HepG2 cell line. This 3D model was later applied to assess the genotoxic effects of polycyclic aromatic hydrocarbons (PAH). In the first experimental part, we monitored the growth, differentiation and metabolic activity during the spheroids’ cultivation. The results demonstrated overall stability of the spheroids during their cultivation, gradual maturation, and peak metabolic activity between days 17 and 22 of the spheroids’ age, confirmed by the expression of hepatic markers (ALB, CK18, HNF4α) and induction of metabolic enzymes (CYP1A1, CYP3A4, NAT2, UGT1A1). Based on these results, 21-day-old spheroids were selected as the optimal model. In the second experimental part, the spheroids were exposed to PAHs: benzo(g)perylene (BGP), benzo(b)fluoranthene (BBF), their mixture, and benzo(a)pyrene (BaP) for 24 and 96 hours. A flow cytometry analysis revealed that BGP increased the P21- and γH2AX-positive cells, whereas BBF and BaP additionally elevated the percentage of GADD45α-positive cells. The mixture did not display synergistic effects. Higher concentrations produced stronger responses, while long-term exposure indicated either activation of repair mechanisms or accumulation of DNA damage, depending on the species of PAH. Based on biomarker expression, the genotoxic potential of PAHs was ranked as following: BGP < BBF < BaP. HepG2 sferoidi in vitro 3D celični model ksenobiotiki genotoksičnost citotoksičnost policiklični aromatski ogljikovodiki pretočna citometrija reverzna transkripcija in verižna reakcija s polimerazo HepG2 spheroids in vitro 3D cell model xenobiotics genotoxicity cytotoxicity polycyclic aromatic hydrocarbons flow cytometry quantitive reverse transcription polymerase chain reaction true false false [V. Ham] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-09-21 07:17:36 2025-09-21 07:17:40 2025-10-12 03:45:50 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 577:576.385(043.2) 265259 249819907 53282.pdf 53282.pdf 1 28169EEFC95EB72F96DF4CD0E140C47F 1154e298137396df6f319521380cbcb978e875b6fc44c79580dbae29da5047b8 1cf427dd-96aa-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=218401 Biotehniška fakulteta 0 0 0