enoviti magistrski študij farmacija
Urša Pratneker (Author), Barbara Ostanek (Mentor), Tilen Kranjc (Co-mentor)

Abstract

Oksidativni stres je stanje v organizmu, ki nastane zaradi prekomerne količine reaktivnih kisikovih zvrsti. Te preko pospešene diferenciacije osteoklastov in zmanjšane diferenciacije in aktivnosti osteoblastov vodijo do povečane izgube kostne mase. Slednja pa je vključena v patogenezo številnih kostnih bolezni. Prav tako je znano, da epigenetski mehanizmi: metilacija DNA, posttranslacijske modifikacije histonov in nekodirajoče RNA pomembno vplivajo na izražanje številnih genov, vključenih v proces kostne prenove. Slabo pa je poznana interakcija med oksidativnim stresom in epigenetskimi mehanizmi v kosteh. Namen našega dela je bil proučiti vpliv treh dejavnikov: oksidativnega stresa, inhibitorja metilacije in antioksidanta na izražanje 8 epigenetskih proteinov (DNMT3A, MBD1, MYST1, HAT1, HDAC6, HDCA9, SIRT1, SIRT6) in njihov vpliv na izražanje genov za RANKL in OPG, v človeških osteosarkomskih celicah, v dveh časovnih točkah – 24 in 72 ur. Ovrednotili smo vpliv vodikovega peroksida in 5-azacitidina, kombinacijo oksidativnega stresa in 5-azaciditidna ter oksidativnega stresa in TEMPOL-a na izražanje proučevanih genov. Izražanje genov smo določali s kvantitativno verižno reakcijo s polimerazo v realnem času. Z uporabo statističnih metod nismo zaznali nobenih signifikantnih razlik v izražanju proučevanih genov. Na osnovi trendov, ki smo jih dobili na grafih, ki prikazujejo izražanje genov pa sklepamo, da oksidativni sodeluje pri uravnavanju epigenetskih mehanizmov. Zaznali smo povečano izražanje MBD1, HAT1, MYST1, SIRT1, SIRT6 in HDAC9, ter znižano izražanje HDAC6. Inhibitor metilacije 5-azacitidin, zmanjša izražanje DNMT3A in poveča izražanje SIRT1, SIRT6 in HDAC6. Antioksidativno delovanje TEMPOL-a se je pokazalo po 24-ih urah, saj se je izražanje genov, katerih izražanje se je pod vplivom oksidativnega stresa bodisi povečalo, bodisi zmanjšalo, v večji meri vrnilo na nivo, kot je le-ta v kontrolnem vzorcu. Pod vplivom omenjenih dejavnikov se spreminja tudi izražanje dveh genov vključenih v triado RANK/RANKL/OPG. Z vodikovim peroksidom izzvan oksidativni stres poveča izražanje RANKL, izražanje OPG pa se ne spremeni. Povečano izražanje mRNA za RANKL smo zaznali tudi pri tretiranju celic s 5-azacitidinom. Naša študija je ena prvih, ki proučuje povezavo oksidativnega stresa in epigenetskih mehanizmov. Predstavlja izhodišče za nadaljnje študije, s katerimi bi lahko dobili boljši vpogled, kako oksidativni stres preko epigenetskih mehanizmov vpliva na kostno homeostazo, kar je pomembno s stališča boljšega razumevanja patogeneze kot tudi razvoja novih učinkovin za zdravljenje kostnih bolezni.

Keywords

epigenetika;oksidativni stres;osteosarkomske celice;celična linija;epigenetski mehanizmi;celice HOS;Osteoporoza;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FFA - Faculty of Pharmacy
Publisher: [U. Pratneker]
UDC: 616.71-006+616.71-007.234(043.3)
COBISS: 4458353 Link will open in a new window
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Other data

Secondary language: English
Secondary title: ǂThe ǂinfluence of oxidative stress on expression of genes invloved in epigenetic mechanisms in human osteosarcoma cells
Secondary abstract: Excessive amount of reactive oxigen species in organism leads to a cellular state known as oxidative stress. They stimulates osteoclasts differentiation and inhibit differentiation and activation of osteoblasts, which leads to bone resorption and several bone diseases. Oxidative stress through epigenetic mechanisms: DNA methylation, histone modifications and non-coding RNAs, alters expression of several genes involved in bone turnover. Interaction between oxidative stress and epigenetic mechanisms is still not completely understood. The aim of our work was to study three factors that could have impact on epigenetic mechanism in human osteosarcoma cells: oxidative stress, inhibitor of DNA methylation and antioxidant. Cells were exposed to hydrogen peroxide, 5-azacitidine, combination of hydrogen peroxide and 5-azacitidine and combination of hydrogen peroxide and TEMPOL, for 24 and 72 hours. Using real-time PCR we monitored expression of 10 genes (DNMT3A, MBD1, MYST1, HAT1, HDAC6, HDCA9, SIRT1, SIRT6, OPG in RANKL) after exposure to certain conditions. We did not prove any significant changes in gene expression. But according to trends shown in graphs, we deduce that oxidative stress influence on epigenetic mechanisms. We observed increase expression of MBD1, HAT1, MYST1, SIRT6, SIRT1 in HDAC9 and decrease expression of HDAC6. 5-azacitidine decreases expression of DNMT3A and increases expression of SIRT1, SIRT6 and HDCA6. The protective effect of antioxidant TEMPOL was observed, TEMPOL alleviated influence of oxidative stress on gene expression after 24 hours treatment. We also observed effects of certain conditions on RANK/RANKL/OPG signalling pathway. RANKL expression was downreglated after treatment with hydrogen peroxide, but there was no changes in OPG expression. Decrease of RANKL expresison was detected after treatment with 5-azacitidine. This was one of the first exsperiments to study the connection between oxidative stress and epigenetic mechanisms. This field is interesting for further investigations, which will help to understand how oxidative stress through epigenetic mechanisms influence on bone homeostasis. This is important for better understanding of patogenesis and development new drug substances in treament of bone diseases.
Secondary keywords: oxidative stress epigenetic mechanisms HOS cell line;
Type (COBISS): Master's thesis/paper
Thesis comment: Univ. Ljubljana, Fak. za farmacijo
Pages: VIII, 58 f.
ID: 12044629