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Efekat mezenhimalnih matičnih ćelija na oštećenje jetre uzrokovano aktivacijom NKT ćelija

Mesenchymal stem cells-mediated modulation of NKT cell-dependent liver injury

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Author
Gazdić, Marina M.
Mentor
Volarević, Vladislav
Committee members
Lukić, Miodrag
Arsenijević, Nebojša
Stojković, Miodrag
Bumbaširević, Vladimir
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Abstract
Mezenhimalne matične ćelije (engl. mesenchymal stem cells, MSCs) se zbog svojih imunomodulatornih karakteristika smatraju potencijalnim terapijskim sredstvom za lečenje fulminantnog hepatitisa. Međutim, efekat MSCs na fenotip i funcije urođenoubilačkih T limfocita (engl. natural killer T cells, NKT), glavnih efektorskih ćelija u ovoj bolesti još uvek nije poznat. Kako bi se ispitao uticaj MSCs na hepatotoksičnost NKT ćelija, u ovom istaživanju su korišćeni modeli akutnog oštećenja jetre koji su C57Bl/6 miševima indukovani aplikacijom konkanavalina A (engl. concanavalin A, ConA) odnosno α-galaktoceramida (engl. α-galactosylceramide, α-GalCer). Biohemijski testovi i kvantitativna histologija su ukazali da MSCs značajno redukuju akutno oštećenje hepatocita izazvano primenom Con A ili α-GalCer-a, što je u korelaciji sa smanjenom zastupljenošću inflamacijskih NKT ćelija (TNF-α-, IFN-γ-, T-bet+ CD4+ i CD1d tetramer+ kao i GATA3+, IL-4+ NKT ćelija) u jetri i smanjenom koncentracijom TNF-α, I...FN-γ i IL-4 u serumu miševa sa hepatitisom. MSCs su suprimirale produkciju TNF-α, IFN-γ i IL-4, a povećavale produkciju imunosupresivnog IL-10 u α-GalCer-om stimulisanim NKT ćelijama jetre, ex vivo. Aplikacija MSCs smanjila je ekspresiju FASL, CD107 i TRAIL na NKT ćelijama jetre, i sledstveno, smanjila citotoksičku aktivnost NKT ćelija prema hepatocitima in vitro. Primena 1-metil triptofana, farmakološkog inhibitora indolamin 2, 3-dioksigenaze (engl. indolamine 2,3-dyoxigenase, IDO) i L-monometil arginin citrata, specifičnog inhibitora inducibilne azot oksid sintaze (engl. inducible nitric oxide synthase, iNOS) ukinula je hepatoprotektivni efekat kondicioniranog medijuma generisanog od MSCs, kao i imunomodulatorno dejstvo MSCs na NKT ćelije in vitro. IDO i iNOS su glavni medijatori kojim su humane MSCs suprimirale efektorske funkcije α-GalCer-om stimulisanih humanih mononuklearnih ćelija periferne krvi. Najvažniji zaključak ove disertacije je da MSCs katalitičkom aktivnošću enzima IDO i iNOS, suprimiraju citotoksičnost i produkciju inflamacijskih citokina u NKT ćelijama u jetri i redukuju oštećenje hepatocita.

Mesenchymal stem cells (MSCs) are, due to immunomodulatory characteristics, considered as novel agents in the treatment of immune-mediated acute liver failure. Howeer, the effects of MSCs on phenotype and function of natural killer T (NKT) cells, major effector cells in fulminant hepatitis, is not understood. We used concanavalin A (ConA) - and α-galactosylceramide (α-GalCer)-induced liver injury to evaluate effects of MSCs on NKT-dependent hepatotoxicity. Mouse MSCs (mMSCs) significantly reduced Con A- and α-GalCer-mediated hepatitis in C57Bl/6 mice, as demonstrated by histopathological and biochemical analysis, attenuated influx of inflammatory (T-bet+ TNF-α, IFN-γ producing and GATA3+, IL-4-producing) liver NKT cells and down-regulated TNF-α, IFN-γ and IL-4 levels in the sera. The liver NKT cells cultured in vitro with mMSCs produced lower amounts of inflammatory cytokines (TNF-α, IFN-γ, IL-4) and higher amounts of immunosuppressive IL-10 upon α-GalCer stimulation. mMSC treatment at...tenuated expression of apoptosis-inducing ligands (FASL, CD107 and TRAIL) on liver NKT cells and suppressed the expression of pro-apoptotic genes in the livers of α-GalCer-treated mice. mMSCs reduced cytotoxicity of liver NKT cells against hepatocytes in vitro. The presence of 1-methyl-DL-tryptophan, a specific inhibitor of indoleamine 2,3-dioxygenase (IDO) or L-NG-monomethyl Arginine citrate, specific inhibitor of inducible nitric oxide synthase (iNOS), in mMSC-conditioned medium injected to α-GalCer-treated mice, counteracted the hepatoprotective effect of mMSCs in vivo, and restored pro-inflammatory cytokine production and cytotoxicity of NKT cells in vitro. Human MSCs in iNOS and IDO-dependent manner, attenuated the production of inflammatory cytokines in α-GalCer-stimulated human peripheral blood mononuclear cells and reduced their cytotoxicity against HepG2 cells. In conclusion, MSCs protect from acute liver injury by attenuating cytotoxicity and capacity of liver NKT cells to produce inflammatory cytokines in iNOS and IDO dependent manner.

Faculty:
Универзитет у Крагујевцу, Факултет медицинских наука
Date:
15-12-2017
Keywords:
Mezenhimalne matične ćelije / Mesenchymal stem cells / NKT ćelije / imunosupresija / akutno oštećenje jetre / NKT cells / Immunosuppression / Acute liver injury
[ Google Scholar ]
Handle
https://hdl.handle.net/21.15107/rcub_nardus_9192
URI
http://eteze.kg.ac.rs/application/showtheses?thesesId=5547
https://nardus.mpn.gov.rs/handle/123456789/9192
https://fedorakg.kg.ac.rs/fedora/get/o:905/bdef:Content/download

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