20.500.12556/RUL-188567 Vpliv mutiranega keratina 14 na adhezijo keratinocitov Effect of mutant keratin 14 on keratinocyte adhesion Keratini (K) so proteini intermediarnih filamentov, ki v keratinocitih oblikujejo keratinski citoskelet, ki zagotavlja mehansko stabilnost epidermisa kože. V bazalnih celicah epidermisa se izražata K14 in K5, ki skupaj tvorita heterodimere. Mutacije v genu za K14 so povezane z nastankom bulozne epidermolize simpleks (EBS), za katero je značilna povečana krhkost kože in nastanek mehurjev. Mutacija R125P v genu za K14 povzroča spremembe v organizaciji keratinskega citoskeleta, lahko pa vpliva tudi na adhezijo keratinocitov in spremenjeno izražanje adhezijskih proteinov. Namen magistrske naloge je bil ovrednotiti vpliv mutacije R125P in njenega deleža na adhezijo keratinocitov ter izražanje in lokalizacijo adhezijskih proteinov. Prav tako smo želeli ovrednotiti vpliv količine mutiranega keratina 14 na organizacijo keratinskega citoskeleta v keratinocitih. Za raziskavo smo uporabili celične linije NEB-1, ki so izražale konstrukte EGFP-K14 divjega tipa ali EGFP-K14 R125P v različnih deležih. Z metodo SDS-PAGE in prenosom po Westernu smo ovrednotili izražanje izbranih adhezijskih proteinov, z imunofluorescenčnim mikroskopiranjem njihovo lokalizacijo, s testi adhezije pa sposobnost pritrjevanja celic na različne podlage. Z imunofluorescenčnim označevanjem K5 smo ocenili vpliv mutacije K14 na organizacijo keratinskega citoskeleta. Za analizo organizacije tkiva in izražanja adhezijskih proteinov smo pripravili tudi tkivne modele kože. Ugotovili smo, da različni deleži mutiranega K14 povzročijo različne spremembe v izražanju in lokalizaciji adhezijskih proteinov ter v sposobnosti adhezije keratinocitov. Dodatek komponente zunajceličnega matriksa je izboljšal adhezijo celic z 25% deležem mutiranega K14, kar nakazuje možnost reševanja adhezijskega fenotipa te celične linije. Ugotovili smo tudi, da se organizacija keratinskega citoskeleta spreminja, glede na količino izraženega EGFP-K14 R125P konstrukta. Keratin (K) proteins are intermediate filament proteins that form the keratin cytoskeleton in keratinocytes, providing mechanical stability to the epidermis of the skin. In basal epidermal cells, K14 and K5 are expressed and form heterodimers. Mutations in the K14 gene are associated with the development of epidermolysis bullosa simplex (EBS), a disorder characterized by increased skin fragility and blister formation. The R125P mutation in the K14 gene causes alterations in the organization of the keratin cytoskeleton and may also affect keratinocyte adhesion and the expression of adhesion proteins. The aim of this master’s thesis was to evaluate the effect of the R125P mutation and its proportion on keratinocyte adhesion, as well as on the expression and localization of adhesion proteins. We also aimed to evaluate the effect of the amount of mutant keratin 14 on the organization of the keratin cytoskeleton in keratinocytes. For this study, we used NEB-1 cell lines expressing either EGFP-K14 wild-type or EGFP-K14 R125P constructs at different proportions. The expression of selected adhesion proteins was evaluated using SDS-PAGE followed by Western blotting, while their localization was assessed by immunofluorescence microscopy. Cell adhesion assays were performed to evaluate the ability of the cells to adhere to different substrates. Immunofluorescent labeling of K5 was used to assess the effect of the K14 mutation on the organization of the keratin cytoskeleton. Tissue models of skin were also prepared to analyze tissue organization and the expression of adhesion proteins. We found that different proportions of mutant K14 resulted in distinct changes in the expression and localization of adhesion proteins, as well as in the adhesive capacity of keratinocytes. The addition of an extracellular matrix component improved cell adhesion in the cell line containing 25% mutant K14, suggesting a potential rescue of the adhesion phenotype in this cell line. We also found that the organization of the keratin cytoskeleton varied depending on the amount of the expressed EGFP-K14 R125P construct. Keratin 14 keratinociti koža bulozna epidermoliza simpleks adhezija keratinocitov proteini adhezoma tkivni model kože Keratin 14 keratinocytes skin epidermolysis bullosa simplex keratinocyte adhesion adhesion proteins skin tissue model true false false [V. Klepec] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-09-24 08:37:06 2026-09-24 08:37:11 2026-09-25 10:09:11 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 547.962.9(043.2) 292240 292480003 62773.pdf 62773.pdf 1 D1E7D43E5922137E99EE1654AB86AA21 61ff25d3723c09f7e73812a5103fa5b4552192f078ffeadf37cf23c1cb0b8c3e 4d9cea82-b7e2-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=248817 Biotehniška fakulteta 0 0 0