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Genska ekspresija enzima antioksidativne zaštite u tkivima pacova i humanoj krvi : uticaj spoljnih faktora i korelacija sa biohemijskim markerima oksidativnog stresa

Gene expression analyses of antioxidative enzymes in rats' tissues and human blood: the influence of external factors and correlation with biochemical markers of oxidative sress

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2014
Disertacija.pdf (8.305Mb)
Author
Ninić, Ana R.
Mentor
Spasojević-Kalimanovska, Vesna
Committee members
Spasić, Slavica
Bogavac-Stanojević, Nataša
Kotur-Stevuljević, Jelena
Joiksić, Gordana
Metadata
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Abstract
Genska ekspresija je proces koji obuhvata transkripciju gena, obradu transkripta i njegovu translaciju u protein. Kvantifikacijom genske ekspresije može se videti da li dolazi do ushodne ili nishodne regulacije specifičnih gena i posledica takve regulacije. Značajnu ulogu u zaštiti od oksidativnog stresa (prekomerne količine stvorenih slobodnih radikala) u intra- i ekstraćelijskom prostoru imaju enzimi antioksidativne zaštite, superoksid-dismutaza (SOD) i paraoksonaza (PON). Interakcije gena antioksidativne zaštite i fizičke aktivnosti, gena i tipa dijete, gena i terapije hipolipemicima mogu da modifikuju ekspresiju tih gena; fizička aktivnost slobodnim radikalima, aterogena dijeta i terapija atorvastatinom svojim komponentama, direktno ili indirektno interagujući sa transkripcionim faktorima. Cilj istraživanja je bio da se ispita uticaj fizičke aktivnosti na gensku ekspresiju izoenzima SOD (Cu/Zn SOD i Mn SOD) u limfocitima sportista i uticaj aterogene dijete i terapije atorvastatinom... na gensku ekspresiju izoenzima PON (PON1, PON2, PON3) u jetri, belom masnom tkivu i aorti eksperimentalnih životinja. Pored toga, cilj je bio i da se uporede metode za izolaciju RNK iz krvi i ispita njihov uticaj na vrednosti genske ekspresije izoenzima SOD. Istraživanje je obuhvatilo odrasle osobe (kontrolna grupa), grupu fudbalera pre i nakon 12 nedelja intenzivnog treniranja i eksperimentalne životinje koje su podeljene u 3 grupe (1. grupa na normalnoj dijeti – ND grupa, 2. grupa na aterogenoj dijeti - AD i 3. grupa na aterogenoj dijeti i terapiji atorvastatinom - ADA grupa). RNK je izolovana TRIzol™ metodom (TRI) iz mononuklearnih ćelija periferne krvi (TRIMĆPK) i na aparatu ABI Prism™ 6100 Nucleic Acid PrepStation (ABI) iz krvi (ABIKRV) i mononuklearnih ćelija periferne krvi (ABIMĆPK). Takođe, RNK je izolovana pomoću TRIzol™ metode iz ispitivanih organa pacova. Za kvantifikaciju relativne genske ekspresije korišćena je Real time PCR metoda. Fudbaleri su nakon 12 nedelja treniranja bili u oksidativnom stresu. Vrednosti genske ekspresije Cu/Zn SOD se nisu statistički značajno promenile (p=0,106), dok su vrednosti Mn SOD bile značajno veće (p=0,031) nakon treniranja. Vrednosti genske ekspresije Cu/Zn SOD su bile parametar koji nezavisno utiče na ukupnu aktivnost SOD u plazmi (p=0,025), a koncentracija tiobarbituratna kiselina-reagujućih supstanci je imala ulogu prediktivnog faktora (p=0,017) za vrednosti genske ekspresije Mn SOD...

Gene expression is a process that includes transcription, transcript processing and its translation into protein. Gene expression quantification can be used to see weather specific genes are up or down regulated and consequences of such regulation. Enzymes of antioxidative defense system, superoxide dismutase (SOD) and paraoxonase (PON) have important roles in protection against oxidative stress (excessive amount of generated free radicals) in intra- and extracellular space. Interaction of antioxidative defense genes and physical activity, genes and type of diet, genes and hipolipemic drugs can modify the expression patterns of these genes; physical activity by free radicals formation, atherogenic diet and atorvastatin therapy with its components, directly or indirectly interacting with transcription factors. The aim of this study was to investigate the influence of physical activity on SOD isoenzymes’ (Cu/Zn SOD and Mn SOD) gene expression in sportsmen’ lymphocytes and the influence o...f atherogenic diet and atorvastatin therapy on paraoxonase isoenzymes’ (PON1, PON2 and PON3) gene expression in liver, white adipose tissue and aorta of experimental animals. In addition, we compared methods for RNA isolation from blood and examined their influence on SOD isoenzymes’ gene expression. The study included control group, soccer players before and after 12 weeks of intensive trainings and rats who were divided into 3 groups (1. group on normal diet - ND group, 2. group on atherogenic diet - AD group and 3. group on atherogenic diet with atorvastatin therapy- ADA group). RNA was isolated using Trizol™ method (TRI) from peripheral blood mononuclear cells (TRIPBMC) and using ABI Prism ™ 6100 Nucleic Acid PrepStation (ABI) from blood (TRIBLOOD) and from peripheral blood mononuclear cells (ABIPBMC). RNA was also isolated from rats’ organs using TRI method. Real time PCR was used for relative gene expression quantification. Soccer players were in oxidative stress after 12 weeks of intensive trainings. Cu/Zn SOD gene expression levels did not change significantly after training (p=0.106), while those of Mn SOD were significantly higher (p=0.031) after than before training. Cu/Zn SOD expression levels were independent parameter that influenced total SOD activity in plasma (p=0.025). Concentration of thiobarbituric acid reactive substances was predictive parameter for Mn SOD gene expression values (p=0.017)...

Faculty:
Универзитет у Београду, Фармацеутски факултет
Date:
20-02-2014
Projects:
  • Interactive role of dyslipidemia, oxidative stress and inflammation in atherosclerosis and other diseases: genetic and biochemical markers (RS-175035)
Keywords:
genska ekspresija / gene expression / superoksid-dismutaza / paraoksonaza / oksidativni stres / metoda izolacije RNK / superoxide dismutase / paraoxonase / oxidative stress / RNA isolation method
[ Google Scholar ]
Handle
https://hdl.handle.net/21.15107/rcub_nardus_3161
URI
http://eteze.bg.ac.rs/application/showtheses?thesesId=1364
https://nardus.mpn.gov.rs/handle/123456789/3161
https://fedorabg.bg.ac.rs/fedora/get/o:8489/bdef:Content/download
http://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=45053967

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