20.500.12556/RUL-187685 Preskušanje plazmidov za izražanje toksinov namenjenih cepljenju proti raku Functionality testing of toxin-encoding plasmids for cancer vaccination Začetki imunoterapije raka segajo v konec 19. stoletja, ko je William B. Coley uvedel pristop intratumorskega injiciranja s toploto inaktiviranih bakterij, ki proizvajajo toksine. Ena najbolj obetavnih imunoterapevtskih strategij je cepljenje in situ, ki se opira na lokalno ablacijo tumorja, da sproži tumorsko specifičen imunski odziv s sproščanjem tumorskih antigenov iz umirajočih rakavih celic. V sklopu magistrske naloge smo ovrednotili osnovno funkcionalnost plazmidov z zapisom za bakterijske toksine, namenjenih in situ protitumorskem cepljenju z genskim elektroprenosom (GEP, angl. gene electrotransfer). Z uporabo reporterskega sistema z izboljšanim zelenim fluorescenčnim proteinom (EGFP, angl. enhanced green fluorescent protein) smo primerjali učinkovitost sekrecijskih signalov iz genov za interlevkin 2 in perforin 1 ter določali izražanje plazmidov za izražanje porotvornih toksinov in superantigenih toksinov. Plazmide smo transfecirali v celično linijo mišjega melanoma B16F10, izražanje EGFP smo določali s slikanjem fluorescence, izražanje toksinov s kvantitativnim PCR, citotoksičnost pa s testom PrestoBlueTM. Rezultati so pokazali učinkovito sekrecijo pri konstruktu s sekrecijskim signalom PRF1 ter uspešno transkripcijo vseh analiziranih toksinov v transfeciranih celicah. Ugotovitve potrjujejo primernost pripravljenih plazmidov za nadaljnje raziskave terapevtske učinkovitosti pri in situ protitumorskem cepljenju. The origins of cancer immunotherapy date back to the late 19th century, when William B. Coley introduced the approach of intratumoral injection with heat-inactivated toxin-producing bacteria. One of the most promising immunotherapeutic strategies is in situ vaccination, which relies on local tumor ablation to trigger a tumor-specific immune response by releasing tumor antigens from dying cancer cells. As part of this master's thesis, we evaluated the basic functionality of plasmids encoding bacterial toxins as potential candidates for in situ antitumor vaccination by gene electrotransfer (GEP). Using an EGFP (enhanced green fluorescent protein) reporter system, we compared the efficiency of secretion signals from the interleukin 2 and perforin 1 genes and determined the expression of plasmids for the expression of pore-forming toxins and superantigenic toxins. The plasmid was transfected into the B16F10 mouse melanoma cell line, and EGFP expression was determined by fluorescence imaging, toxin expression by quantitative PCR, and cytotoxicity by the PrestoBlueTM assay. The results showed efficient secretion in the construct with the PRF1 secretion signal and successful transcription of all analyzed toxins in transfected cells. The findings confirm the suitability of the prepared plasmids for further studies of therapeutic efficacy in in situ antitumor vaccination. in situ cepljenje genski elektro prenos plazmid tvorba por toksin in situ vaccination gene electrotransfer plasmid pore-forming toxin true false false [K. Lovišček] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-09-13 07:17:04 2026-09-13 07:17:10 2026-09-15 03:48:13 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 615.37:602.68:577.27 289008 290906883 61656.pdf 61656.pdf 1 44EC0A8B55A8AAEA6463084A43DA3E4D 9694f07d31947907ee8dd5c778990d9a7e772b424b3eb2abb140afd3f816b4dd 577b104c-af32-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=247387 Biotehniška fakulteta 0 0 0