magistrsko delo
Povzetek
Kolagen VII je protein zunajceličnega matriksa (ECM), kodiran z genom COL7A1. Tvori sidrne fibrile, ki se raztezajo od dna bazalne lamine do papilarnega dermisa, in zagotavljajo integriteto dermalno-epidermalnega stika. Mutacije v COL7A1 vodijo do pojava distrofične bulozne epidermolize (DEB). Gre za bolezen krhkosti kože, katere glavni klinični znak je tvorba mehurjev. V magistrskem delu nas je zanimala sestava adhesoma linij iPSC, pridobljenih z reprogramiranjem fibroblastov iz bolnika z dominantno (DDEB) in bolnika z recesivno (RDEB) DEB. Analiza z masno spektrometrijo je pokazala nepričakovane razlike med adhesomoma, ki so se kazale v obliki sestave in količine proteinov. Po pričakovanjih se kolagen VII ni izražal pri nobeni od celičnih linij. V obeh vzorcih so bili prisotni številni proteini, povezani s citoskeletom, ECM in adhezijo. Zabeležili smo prisotnost večine proteinov konsenzusnega adhesoma, retikularnega adhesoma in klatrinskega interaktoma. Z masno spektrometrijo smo analizirali tudi adhesom netretiranih in z ZnCl2 tretiranih linij fibroblastov, pridobljenih iz bolnika z DDEB in bolnika z RDEB ter iz zdrave osebe (WT). Sestava in količina proteinov sta bili odvisni tako od celične linije kot od tega, ali so bile celice tretirane z ZnCl2. Tudi tu smo pri vseh vzorcih zabeležili prisotnost številnih proteinov, povezanih s citoskeletom, ECM in adhezijo. Tretiranje celic z ZnCl2 je pri linijah, pridobljenih iz bolnikov, povečalo izražanje prej omenjenih proteinov, kar kaže na pozitiven učinek te kemikalije in njeno potencialno uporabnost za razvoj zdravil. Celično linijo iPSC, pridobljeno iz bolnika z DDEB, smo uspešno diferencirali v fibroblaste in keratinocite. Fibroblastom podobne celice so izražale vimentin, marker za fibroblaste, keratinocitom podobne celice pa keratin 14, marker za bazalne keratinocite. S pomočjo metabolnih testov MTS smo preizkusili učinek ZnCl2, lizinoprila, trisa in niacinamida na celične linije fibroblastov. Ugotovili smo, da je učinek odvisen od kemikalije, njene koncentracije, časa tretiranja in celične linije. Največji pozitiven učinek na celice smo zabeležili po 72 urah. Višje koncentracije kemikalij so bile citotoksične.
Ključne besede
adhesom;kolagen VII;dominantna distrofična bulozna epidermoliza;recesivna distrofična bulozna epidermoliza;inducirane pluripotentne matične celice;citoskelet;zunajcelični matriks;masna spektrometrija;
Podatki
Jezik: |
Slovenski jezik |
Leto izida: |
2024 |
Tipologija: |
2.09 - Magistrsko delo |
Organizacija: |
UL BF - Biotehniška fakulteta |
Založnik: |
[T. Fabčič] |
UDK: |
616.529.1(043.2) |
COBISS: |
194150915
|
Št. ogledov: |
28 |
Št. prenosov: |
4 |
Ocena: |
0 (0 glasov) |
Metapodatki: |
|
Ostali podatki
Sekundarni jezik: |
Angleški jezik |
Sekundarni naslov: |
Adhesome of iPSC lines, derived from a patient with dominant and a patient with recessive dystrophic epidermolysis bullosa |
Sekundarni povzetek: |
Type VII collagen is an extracellular matrix (ECM) protein encoded by the COL7A1 gene. Anchoring fibrils formed by collagen VII extend from the basement membrane to the papillary dermis, providing integrity to the dermo-epidermal junction. Mutations in COL7A1 lead to the occurrence of dystrophic epidermolysis bullosa (DEB), a skin fragility disorder, characterized by blistering of the skin. In this master's thesis, we were interested in adhesome composition of iPSC lines, obtained by reprogramming fibroblasts from a patient with dominant (DDEB) and a patient with recessive (RDEB) DEB. Analysis by mass spectrometry revealed unexpected differences between the adhesomes, which were manifested in the form of protein composition and quantity. As expected, type VII collagen was not expressed in either of the two iPSC lines. A number of cytoskeletal, ECM, and adhesion-related proteins were present in both samples. We recorded the presence of most proteins of the consensus adhesome, reticular adhesome and clathrin interactome. Adhesome of untreated and with ZnCl2 treated fibroblast lines, obtained from a patient with DDEB and a patient with RDEB and from a healthy person (WT) were also analyzed by mass spectrometry. Protein composition and quantity were dependent on both the cell line and whether the cells were treated with ZnCl2. A number of cytoskeletal, ECM, and adhesion-related proteins were present in all samples. Treatment of cells with ZnCl2 increased the expression of the mentioned proteins in both cell lines, derived from the patients, which indicates the positive effect of this chemical and its potential utility for drug development. The iPSC line derived from a patient with DDEB was successfully differentiated into fibroblats and keratinocytes. Fibroblast-like cells expressed vimentin, a fibroblast cell marker and keratinocyte-like cells expressed keratin 14, a marker of basal keratinocytes. The effect of ZnCl2, lisinopril, tris and niacinamide on fibroblast cell lines was tested using MTS assay. We found that the effect depends on the chemical, its concentration, treatment time and cell line. The greatest positive effect on the cells was recorded after 72 hours. Higher concentrations of chemicals were found to be cytotoxic. |
Sekundarne ključne besede: |
adhesome;type VII collagen;dominant dystrophic epidermolysis bullosa;recessive dystrophic epidermolysis bullosa;induced pluripotent stem cells;cytoskeleton;extracellular matrix;mass spectrometry; |
Vrsta dela (COBISS): |
Magistrsko delo/naloga |
Študijski program: |
0 |
Konec prepovedi (OpenAIRE): |
1970-01-01 |
Komentar na gradivo: |
Univ. v Ljubljani, Biotehniška fak., Oddelek za biologijo, Magistrsko delo magistrskega študijskega programa 2. stopnje Molekulska in funkcionalna biologija |
Strani: |
1 spletni vir (1 datoteka PDF (XII, 79 str.)) |
ID: |
23530173 |