20.500.12556/RUL-161711 Ravni izražanja in aktivnosti katepsina X in izooblik enolaze v fenotipsko različnih populacijah celic SH-SY5Y Expression and activity levels of cathepsin X and enolase isoforms in phenotypically distinct populations of SH-SY5Y cells Osnovna gradbena enota centralnega živčnega sistema je nevron. Za preučevanje procesov v nevronih se uporabljajo številni in vitro celični modeli in med njimi tudi diferencirane celice SH-SY5Y. Pomembno vlogo pri diferenciaciji nevronov imajo nevrotrofični dejavniki, katerim podobno delovanje izkazuje tudi glikolitični encim γ-enolaza. Nevrotrofično aktivnost γ-enolaze uravnava cisteinska peptidaza katepsin X s proteolitično cepitvijo zadnjih dveh aminokislin na C-koncu γ-enolaze. V sklopu magistrske naloge smo določili ravni izražanja in aktivnosti katepsina X ter izooblik enolaze v fenotipsko različnih populacijah celic SH-SY5Y, katere sestavljajo trije različni fenotipi, in sicer epiteljiski S-tip, nevronski N-tip in vmesni I-tip celic SH-SY5Y. Najprej smo vzpostavili protokol za diferenciacijo in uspešno obogatitev posameznih populacij celic SH-SY5Y, pri čemer smo spreminjali podlago, sestavo gojišča in čas gojenja celic, kar smo spremljali s pomočjo svetlobnega invertnega mikroskopa. 10-dnevno gojenje celic v gojišču z zmanjšano vsebnostjo seruma ob dodani 5 µM retinojski kislini se je izkazalo za uspešno za pridobitev obogatenih populacij celic tipa S in N. Z vrednotenjema izražanja označevalcev s pomočjo prenosa western in konfokalne mikroskopije smo opazili, da je izražanje označevalca epitelijskih celic vimentina povišano v populaciji celic tipa S ter izražanje označevalcev nevronov, in sicer tirozin hidroksilaze in z rastjo povezan protein 43, povišano v populaciji celic tipa N. Na obogatenih populacijah S- in N-tipa smo preučili raven izražanja katepsina X in njegovo aktivnost kot tudi raven izražanje tarče katepsina X v nevronskih celicah, enolaze. Pokazali smo, da je največje izražanje proteina katepsina X v celicah tipa N, medtem ko je bila največja aktivnost katepsina X v celicah tipa S. Izražanje izooblike α-enolaze je neodvisno od posamezne populacije celic SH-SY5Y, medtem ko je višja raven izooblike γ-enolaze značilna za N-tip celic SH-SY5Y, kar smo vrednotili s prenosom western in encimsko-imunskim testom. Izražanje aktivne oblike γ-enolaze je bilo povišano v obeh populacijah celic SH-SY5Y napram kontrolnim celicam, vendar je bilo povišanje izrazitejše v N-tipu celic SH-SY5Y. S pomočjo konfokalne mikroskopije smo pokazali močnejšo ko-lokalizacijo γ-enolaze in katepsina X v celicah N-tipa, predvsem v izrastkih celic, t.i. nevritih, kar nakazuje na vlogo, ki jo ima γ-enolaza v procesu rasti in diferenciacije nevronov ter njeno uravnavanje s katepsinom X. The basic structure of the central nervous system is the neuron. Numerous in vitro cellular models are used to study processes in neurons, including differentiated SH-SY5Y cells. Neurotrophic factors play an important role in the differentiation of neurons, and the glycolytic enzyme γ-enolase also shows similar activity. The neurotrophic activity of γ-enolase is regulated by the cysteine peptidase cathepsin X, which proteolytically cleaves the last two amino acids from the C-terminal domain of γ-enolase. In this master's thesis, we determined the levels of protein expression and activity of cathepsin X and protein expression of enolase isoforms in phenotypically different populations of SH-SY5Y cells, which consist of three distinct phenotypes: epithelial S-type, neuronal N-type, and intermediate I-type SH-SY5Y cells. We first established a protocol for the differentiation and successful enrichment of individual populations of SH-SY5Y cells by modifying the cell culture plate coating, culture medium composition and cell growth time, which we monitored using a light inverted microscope. A 10-day cell culture growing in reduced serum medium content and the addition of 5 µM retinoic acid was found to be successful in obtaining enriched populations of S- and N-type cells. Through the assessment of marker expression using Western blotting and confocal microscopy, we observed that the expression of the epithelial cell marker vimentin was elevated in the S-type cell population, while the expression of neuronal markers, specifically tyrosine hydroxylase and growth-associated protein 43, was elevated in the N-type cell population. In the enriched populations of S- and N-type cells, we examined the expression levels of cathepsin X and its activity, as well as the expression levels of the target of cathepsin X in neuronal cells, enolase. We demonstrated that the highest expression of the cathepsin X protein was found in N-type cells, while the highest activity of cathepsin X was observed in S-type cells. The expression of the α-enolase isoform was independent of the individual SH-SY5Y cell populations, while a higher level of the γ-enolase isoform was characteristic of N-type SH-SY5Y cells, as evaluated by Western blotting and enzyme-linked immunosorbent assay. The expression of the active form of γ-enolase was elevated in both populations of SH-SY5Y cells compared to the control cells, nevertheless the increase was more noticeable in N-type SH-SY5Y cells. Using confocal microscopy, we demonstrated stronger co-localization of γ-enolase and cathepsin X in N-type cells, particularly in the cell extensions, so-called neurites, which suggests a role for γ-enolase in the process of neuronal growth and differentiation, and its regulation by cathepsin X. celice SH-SY5Y diferenciacija enolaza nevrotrofična aktivnost katepsin X SH-SY5Y cells differentiation enolase neurotrophic activity cathepsin X true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2024-09-13 08:45:03 2024-09-13 08:45:11 2024-09-15 03:40:06 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 108091 23601.pdf 23601.pdf 1 44EA7655B674A2874846C3BCF44FC3DB 6d7a26c9c443bba1befedf619f73c914fd9cf9de1447aa6f5705e2ec6377a2e5 ab32afb1-719b-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=190689 Fakulteta za farmacijo 0 0 0