20.500.12556/DKUM-100202 Optimizacija transfekcije ekspresijskih plazmidov v celično linijo HMEC-1 Optimization of transfection of expression plasmids into the HMEC-1 cell line Stalna celična linija HMEC-1 (Human Microvascular Endothelial Cells-1) predstavlja pomemben in vitro celični model za proučevanje delovanja človeškega mikrovaskularnega endotelija. V primerjavi s primarnimi celicami ponuja daljšo življenjsko dobo, manjšo biološko variabilnost ter boljšo ponovljivost poskusov, pri čemer ohranja ključne endotelijske fenotipske značilnosti. Za funkcionalne raziskave in genske manipulacije celične linije HMEC-1 je ključnega pomena doseganje učinkovite transfekcije. Ker HMEC-1 spada med celične sisteme, ki jih je težje transfecirati, in ker univerzalen protokol za vse celične linije ne obstaja, smo v okviru tega dela izvedli natančno optimizacijo pogojev transfekcije. V diplomskem delu smo za optimizacijo transfekcije uporabili plazmid pCMV6-AC-GFP ter tri kationske transfekcijske reagente: FuGENE 4K, ViaFect ter Lipofectamine LTX & PLUS. Optimizacijski protokol na 96-jamičnih mikrotitrskih ploščah je potekal v štirih zaporednih korakih, ki so zajemali določitev optimalne gostote nacepljanja celic, optimizacijo razmerja med reagentom in plazmidno DNA, določitev optimalne količine DNA ter optimizacijo časa inkubacije transfekcijske mešanice. Uspešnost transfekcije in število celic smo ocenjevali z avtomatizirano imunofluorescenčno mikroskopijo na sistemu Cytation 5 po barvanju z barviloma Kalcein AM in Hoechst 33342. Z merjenjem fluorescenčnega signala smo ugotovili, da je bila optimalna gostota nacepljanja 25.000 celic na jamico, saj pri višjih gostotah konfluenca ni več naraščala. Pri optimizaciji parametrov se je reagent FuGENE 4K izkazal za najboljšega pri razmerju 4:1, količini 100 ng DNA in času inkubacije 15 minut. Lipofectamine LTX & PLUS je dosegel največjo učinkovitost pri razmerju 0,4:1, 100 ng DNA in 5 minutah inkubacije. Reagent ViaFect pa je izkazal najvišjo skupno učinkovitost transfekcije pri razmerju 6:1, količini 100 ng DNA ter 10 minutah inkubacije. Spremljanje v časovnih intervalih ter uporaba negativnih kontrol sta potrdili stabilnost izražanja signala. Z merjenjem metabolne aktivnosti z Alamar modrim smo ugotovili, da je reagent FuGENE izkazal precejšnjo citotoksičnost, medtem ko sta ViaFect in Lipofectamine ohranila visoko viabilnost celic. Naš zaključek je, da reagent ViaFect ob uporabi optimiziranih pogojev predstavlja najučinkovitejši sistem za transfekcijo linije HMEC-1. The immortalized cell line HMEC-1 (Human Microvascular Endothelial Cells-1) represents an important in vitro cell model for studying the functions of the human microvascular endothelium. Compared to primary cells, it offers an extended lifespan, reduced biological variability, and improved experimental reproducibility while maintaining key endothelial phenotypic characteristics. Achieving efficient transfection is of critical importance for functional studies and genetic manipulations of the HMEC-1 cell line. Since HMEC-1 is considered a difficult-to-transfect cell model, and a universal protocol applicable to all cell lines does not exist, a precise optimization of transfection conditions was performed in this work. In this diploma thesis, the plasmid pCMV6-AC-GFP and three cationic transfection reagents: FuGENE 4K, ViaFect, and Lipofectamine LTX & PLUS, were utilized for transfection optimization. The optimization protocol on 96-well microtiter plates was conducted in four sequential steps: determining the optimal cell seeding density, optimizing the reagent-to-plasmid DNA ratio, determining the optimal amount of DNA, and optimizing the incubation time of the transfection complex. Transfection efficiency and cell counts were evaluated using automated immunofluorescence microscopy on a Cytation 5 system following staining with Kalcein AM and Hoechst 33342 dyes. By measuring the fluorescence signal, we determined that the optimal seeding density was 25,000 cells per well, as confluence no longer increased at higher densities. During parameter optimization, FuGENE 4K demonstrated peak performance at a 4:1 ratio, 100 ng DNA, and a 15-minute incubation time. Lipofectamine LTX & PLUS achieved its maximum efficiency at a 0.4:1 ratio, 100 ng DNA, and a 5-minute incubation time. The ViaFect reagent exhibited the highest overall transfection efficiency at a 6:1 ratio, 100 ng DNA, and a 10-minute incubation time. Time-course monitoring and the use of negative controls confirmed stability of signal expression. By assessing metabolic activity via the Alamar Blue assay, we determined that FuGENE exhibited considerable cytotoxicity, whereas ViaFect and Lipofectamine preserved high cell viability. We conclude that ViaFect, under the optimized conditions, represents the most effective system for the transfection of the HMEC-1 line. HMEC-1 lipofekcija optimizacija transfekcije GFP-plazmid ViaFect FuGENE Lipofectamine LTX&PLUS celična viabilnost HMEC-1 lipofection transfection optimization GFP plasmid ViaFect FuGENE Lipofectamine LTX&PLUS cell viability true true false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2026-09-08 10:52:11 2026-09-28 17:17:02 2026-09-28 17:52:06 0000-00-00 00:00:00 2026 0 Maribor 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 UN_Tevz_Nina_2026.pdf UN_Tevz_Nina_2026.pdf 1 B7E8D1EAA486542EA4C856EAC359DCEC 2d895470-bb22-11f1-a456-00155d0001be https://dk.um.si/Dokument.php?lang=slv&id=213595 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0