doktorska disertacija
Tomislav Šarenac (Author), Dušica Pahor (Mentor), Dušica Pahor (Thesis defence commission member), Ivan Krajnc (Thesis defence commission member), Andraž Stožer (Thesis defence commission member), Marjan Rupnik (Co-mentor)

Abstract

Celjenje roženičnih ran je pogosto omejeno s fibrozo in tvorjenjem brazgotin, ki jih lahko povzroča transformni rastni faktor β (angl. transforming growth factor – TGF). Nadzirana fibroza, ki jo lahko usmerimo z inzulinu podobnim rastnim faktorjem – 1 (angl. insulin-like growth factor – IGF) in protifibrotičnimi učinkovinami, bi lahko prispevala k ohranjanju prozornosti roženice med celjenjem. S pomočjo stimulacije primarnih človeških keratocitov s TGF-β v brezserumskem gojitvenem mediju smo ustvarili celični model roženične stromalne rane. S slikovno pretočno citometrijo smo analizirali posamezne celice iz celičnih kultur in določali stopnjo nuklearizacije Smad3 in znotrajcelično fluorescenčno intenziteto obarvanega Smad7 in roženičnega Kristalina – aldehidne dehidrogenaze 3A1. Pri preučevanju izločanja proteoglikanov Biglikana in Keratokana v zunajcelični matriks smo uporabili teste ELISA. Skupaj s stimulacijo s TGF-β smo celice obravnavali samo z IGF-1, s suberoilanilidehidroksiamično kislino (SAHA) ali halouginonom; ločenim populacijam smo poleg protifibrotikov dodali še IGF-1. Pri samostojni obravnavi z IGF-1 smo ugotovili zmanjšano translokacijo Smad3 in zvišano količino Aldehidne dehidrogenaze 3A1 znotraj celic. Poleg tega je bilo izločanje proteoglikanov prav tako ugodno za ustvarjanje pogojev prozornosti. SAHA je povzročila zvišanje Smad7 v celicah in inhibirala translokacijo Smad3 v jedra – tudi v kombinaciji z IGF-1. Imunofluorescenčna mikroskopija je pokazala, da je dodatek IGF-1 in v kombinaciji s protifibrotičnimi učinkovinami zavrl transdiferenciacijo v miofibroblaste in spodbudil nastanek fibroblastov. TGF-β/ Smad signalna pot fibroze in zamotnjenosti roženice je bila inhibirana s strani IGF-1; še posebej ob dodatku SAHA kakor tudi s halofuginonom. Zaključujemo, da bi lahko IGF-1 uspešno dodali k zdravljenju s protifibrotičnimi učinkovinami, kar bi omogočilo boljše celjenje roženične rane in tvorbo bolj prozornega tkiva.

Keywords

stroma;lezije;keratociti;fibroblasti;fibroza;brazgotinjenje;rastni faktorji;transformirajoči rastni faktor;insulinu podobni rastni faktor 1;miofibroblasti;celični modeli;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UM - University of Maribor
Publisher: T. Šarenac]
UDC: 617.713-001(043.3)
COBISS: 298261760 Link will open in a new window
Views: 973
Downloads: 116
Average score: 0 (0 votes)
Metadata: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Other data

Secondary language: English
Secondary title: Effect of growth factors and inhibitors of fibrotic healing on keratocytes in cellular models of corneal wound
Secondary abstract: Corneal wound healing is often affected by TGF-β–mediated fibrosis and scar formation. Guided fibrosis with IGF-1 and antifibrotic substances might maintain corneal transparency. Primary human corneal keratocytes under serum-free conditions were used as a model of corneal stromal wounding, with markers of corneal fibrosis and opacity studied under TGF-β2 stimulation. Single-cell imaging flow cytometry was used to determine nuclearization of Smad3, and intracellular fluorescence intensity of Smad7 and the corneal crystallin aldehyde dehydrogenase 3A1. Extracellular matrix proteoglycans keratocan and biglycan were quantified using ELISAs. On the TGF-β2 background, the keratocytes were treated with IGF-1, and suberoylanilidehydroxamic acid (SAHA) or halofuginone with or without IGF-1. IGF-1 alone decreased Smad3 nuclearization and increased aldehyde dehydrogenase 3A1 expression, with favorable extracellular matrix proteoglycan composition. SAHA induced higher Smad7 levels and inhibited translocation of Smad3 to the nucleus, also when combined with IGF-1. Immunofluorescence showed that myofibroblast transdifferentiation is attenuated and appearance of fibroblasts is favored by IGF-1 alone and in combination with the antifibrotic substances. The TGF-β/ Smad pathway of fibrosis and opacity was inhibited by IGF-1, and further with SAHA in particular, and with halofuginone. IGF-1 is thus a valid aid to antifibrotic treatment, with potential for effective and transparent corneal wound healing.
Secondary keywords: Roženica;Disertacije;Rane;Celjenje;
URN: URN:SI:UM:
Type (COBISS): Dissertation
Thesis comment: Univ. v Mariboru, Medicinska fak.
Pages: 63 str.
ID: 10920605