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Micronucleus frequency and polymorphism of GSTT1 and GSTM1 genes in peripheral blood lymphocytes of patients related to the stage of intracervical lesion

dc.contributor.advisorMilošević-Đorđević, Olivera
dc.contributor.otherArsenijević, Slobodan
dc.contributor.otherGrujičić, Darko
dc.contributor.otherJoksić, Gordana
dc.creatorStošić, Ivana
dc.date.accessioned2016-01-05T13:06:32Z
dc.date.available2016-01-05T13:06:32Z
dc.date.available2020-07-03T15:08:14Z
dc.date.issued2015-10-22
dc.identifier.urihttps://nardus.mpn.gov.rs/handle/123456789/3631
dc.identifier.urihttp://eteze.kg.ac.rs/application/showtheses?thesesId=2180
dc.identifier.urihttps://fedorakg.kg.ac.rs/fedora/get/o:564/bdef:Content/download
dc.description.abstractKarcinom grlića materice predstavlja veliki globalni problem, koji je sa 528000 novih slučajeva u 2012. godini predstavljao četvrti oblik maligniteta kod žena u svetu. U Centralnoj Srbiji karcinom grlića materice predstavlja treći oblik maligniteta kod žena. Invazivna forma ovog karcinoma razvija se postepeno kroz itraepitelijalne lezije niskog stepena (low-grade squamous intraepithelial lesion – LSIL) i lezije visokog stepena (high-grade squamous intraepithelial lesion – HSIL). Narušeni hromozomski integritet je karakteristika različitih patoloških stanja. Povećana hromozomska oštećenja, eksprimirana u vidu mikronukleusa (MN), potvrđena je u limfocitima periferne krvi osoba sa dijagnozom kancera. MN predstavljaju ekstranukleusna telašca unutar citoplazme ćelija, a nastaju od acentričnih fragmenata ili celih hromozoma. Prisustvo MN u citoplazmi se povezuje sa hromozomskim oštećenjima i u direktnoj je vezi sa nivoom hromozomskih oštećenja. Kancer je multifaktorijalna bolest i u etiologiji kancera navode se različiti faktori. Povećani rizik za razvoj kancera pripisuje se različitim životnim navikama. Pored egzogenih faktora, genetska osnova može imati ulogu u kancerogenezi. Nulti polimorfizmi dva gena iz GST (Glutathione-S- transferase) familije, GSTT1 i GSTM1 gena, često su analizirani kao potencijalni faktori rizika. Ciljevi ove doktorske disertacije bili su da se utvrdi nivo bazalnog oštećenja naslednog materijala u limfocitima periferne krvi pacijentkinja sa dijagnozom intracervikalnih lezija i zdravih žena kao i korelacija između genetičke nestabilnosti i zdravstvenog stanja, demografskih karakteristika, životnih navika, reproduktivne istorije; procena uticaja polimorfizma GSTT1 i GSTM1 gena u razvoju intracervikalnih lezija, pri čemu je analizom obuhvaćen pojedinačni efekat gena, efekat interakcije analiziranih gena i efekat interakcije gena sa životnim i demografskim karakteristikama. I na kraju, cilj je bio da se utvrdi postojanje korelacije između nivoa bazalnih oštećenja genetičkog oštećenja u limfocitima periferne krvi i genskog profila kod ispitane populacije. Uzorak su činile žene sa dijagnostikovanim intracervikalnim lezijama grlića materice (32 LSIL, 33 HSIL i 39 CC – Ca in situ/Ca invasivum) i 50 zdravih žena. Za utvrđivanje nivoa genetičkih oštećenja u limfocitima periferne krvi pacijentkinja i zdravih kontrola korišćen je citokinezic blok mikronukleus (CBMN) test. Za analizu polimorfizma u GSTT1 i GSTM1 genima primenjena multipleks PCR (Polymerase Chain Reaction) metoda, proizvodi PCR reakcije su razdvojeni na 2% agaroznom gelu u prisustvu SYBR Safe DNA gel stain boje. Svi dobijeni podaci su statistički obrađeni, a vrednosti p<0,05, p<0,01 i p <0,001 su smatrane statistički značajnim. Rezultati doktorske disertacije ukazuju na to da žene sa intracervikalnim lezijama grlića materice imaju veći nivo hromozomskih oštećenja u limfocitima periferne krvi u odnosu na zdrave žene, što je registrovano značajno većom frekvencom MN u limfocitima. Veća zastupljenost binukleusnih (BN) ćelija sa MN zabeležena je u uzorku pacijentkinja sa dijagnozom Ca in situ/Ca invasivum, HSIL i LSIL u odnosu na kontrolu. Žene sa dijagnostikovanim ranim stadijumima lezija, LSIL i HSIL, imale su značajno veće frekvence nukleoplazmatskih mostova (NPM) u odnosu na zdrave žene. Na osnovu rezultata ROC (Receiver Operating Characteristic) analize zaključeno je da je MN u limfocitima periferne krvi dobar biomarker za dijagnostikovanje lezija, a optimalna cut off vrednost iznosila je 10,5 MN/1000 BN sa senzitivnošću 84,2% i specifičnošću 95,1%. Smanjena kinetika limfocita periferne krvi žena sa dijagnostikovanim karcinomom grlića materice registrovana je značajno manjim indeksom nukleusne deobe (NDI) u odnosu na kontrolnu grupu zdravih žena. Uzimajući u obzir stepen lezije, godine starosti, pušenje, spontane i namerne pobačaje, status GSTT1 i GSTM1 gena kao potencijalne modulatore MN frekvence u limfocitima periferne krvi, multipla regresiona analiza na uzorku pacijentkinja, pokazala je da su jedino godine starosti uticale na frekvencu MN. Rezultati iste analize pokazali su da stepen lezije utiče na vrednosti NDI. U uzorku zdravih žena multipla regresiona analiza pokazala je da nijedan od analiziranih faktora nije uticao na MN frekvencu i NDI vrednosti. Na osnovu dobijenih rezultata može se zaključiti da GSTM1 nulti genotip predstavlja faktor rizika za razvoj intracervikalnih lezija. Nosioci GSTM1 nultog genotipa imale su preko 2 puta veći rizik za razvoj lezija. Podelom pacijentkinja na osnovu stepena intracervikalne lezije, rezultati su pokazali značajnu ulogu GSTM1 nultog genotipa samo u razvoju ranih faza lezija (LSIL). Nosioci nultog GSTT1 genotipa nisu imali značajno veći rizik za razvoj intracervikalnih lezija, uzimajući u obzir stadijum lezija, godine starosti i pušenje cigareta.sr
dc.description.abstractCervical cancer is major global problem, and with 528000 new cases in 2012. it represented the fourth form of malignancy in women worldwide. In the region of central Serbia cervical cancer is the third form of malignancy in women. The invasive form of this cancer develops gradually ranging from low-grade squamous intraepithelial lesion - LSIL to high-grade squamous intraepithelial lesion – HSIL. Disturbed chromosomal integrity is the characteristic of various pathological conditions. Increased chromosomal damages expressed in the form of micronuclei (MN) have been confirmed in peripheral blood lymphocytes in persons diagnosed with cancer. MN represents extanuclear small bodies within the cytoplasm of cells and arises from acentric fragments or whole chromosomes. The presence of MN in the cytoplasm is associated with chromosomal defects and is directly related to the level of chromosomal damage. Cancer is a multifactorial disease with diverse factor etiology. Increased risk for cancer development is attributed to the different life style habits. In addition to exogenous factors, genetic background may play a role in carcinogenesis. Null polymorphisms of two genes from GST (Glutathione-S-transferase) family, GSTM1 and GSTT1 genes, are often analyzed as potential risk factors. The aims of this doctoral dissertation were to determine the level of basal damage of the genetic material in peripheral blood lymphocytes of patients with a diagnosis of intracervical lesions and healthy women, as well as the correlation between genetic instability and health status, demographic characteristics, lifestyle habits, reproductive history; assessment of the impact of polymorphism of GSTM1 and GSTT1 genes in the development of intracervical lesions, whereby analysis included the individual effect of genes, the interaction of genes and the effect of the gene interaction with life and demographic characteristics. In addition, the aim was to determine correlation between the level of basal genetic damage in the peripheral blood lymphocytes and gene profiles in the sample population. The study included women diagnosed with intracervical lesions (32 LSIL, HSIL 33 and 39 Ca in situ/Ca invasivum) and 50 healthy women. To determine the level of genetic damage in peripheral blood lymphocytes of patients and healthy controls cytokinesis block micronucleus (CBMN) test was used. For the analysis of polymorphism in GSTT1 and GSTM1 genes multiplex PCR (Polymerase Chain Reaction) method was used; PCR reaction products were separated on 2% agarose gel in the presence of SYBR Safe DNA gel stain. All data were statistically analyzed and the values of p<0.05, p<0.01 and p<0.001 were considered statistically significant. The results of the doctoral dissertation indicate that women with intracervical lesions have a higher level of chromosomal damage in peripheral blood lymphocytes compared to healthy women, which is registered by significantly higher frequency of MN in lymphocytes. Higher distribution of binuclear (BN) cells with MN was observed in the sample of patients diagnosed with Ca in situ/Ca invasivum, HSIL and LSIL compared to the control. Women diagnosed with early stage lesions, LSIL and HSIL had significantly higher frequency of nucleoplasmatic bridges (NPBs) compared to healthy women. Based on the results of ROC (Receiver Operating Characteristic) analysis it was concluded that MN in peripheral blood lymphocytes was a good biomarker for the diagnosis of lesions, and the optimal cut-off value was 10.5 MN/1000 BN with a sensitivity of 84.2% and specificity of 95.1%. Reduced kinetics of peripheral blood lymphocytes of women diagnosed with cervical cancer was registered by significantly lower nuclear division index (NDI) compared to the control group of healthy women. Taking into account degree of lesion, age, smoking, miscarriages and abortions, GSTM1 and GSTT1 status of genes as potential modulators of MN frequency in peripheral blood lymphocytes, multiple regression analysis on a sample of patients showed that the only parameter that affected the MN frequency was age. The results of the same analysis showed that the degree of lesion affected the value of NDI. In a sample of healthy women multiple regression analysis showed that none of the analyzed factors influenced the frequency of MN and NDI values. Based on the obtained results it may be concluded that GSTM1 null genotype represents risk factor for the development of intracervical lesions. Carriers of GSTM1 null genotype had twofold higher risk of developing lesions. By dividing the patients on the basis of the degree of intracervical lesions, the results showed a significant role of GSTM1 null genotype only in the early stages of developing lesions (LSIL). Carriers of null GSTT1 genotype had no significantly higher risk of developing intracervical lesions, taking into account the stage of the lesion, age and cigarette smoking.en
dc.formatapplication/pdf
dc.languagesr
dc.publisherУниверзитет у Крагујевцу, Природно-математички факултетsr
dc.relationinfo:eu-repo/grantAgreement/MESTD/Integrated and Interdisciplinary Research (IIR or III)/41010/RS//
dc.rightsopenAccessen
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/
dc.sourceУниверзитет у Крагујевцуsr
dc.subjectmikronukleussr
dc.subjectmicronucleien
dc.subjectlimfociti periferne krvisr
dc.subjectpolimorfizamsr
dc.subjectGSTT1sr
dc.subjectGSTM1sr
dc.subjectcervikalne intraepitelijalne lezijesr
dc.subjectperipheral blood lymphocytesen
dc.subjectpolymorphismen
dc.subjectGSTT1en
dc.subjectGSTM1en
dc.subjectcervical intraepithelial lesionen
dc.titleFrekvenca mikronukleusa i polimorfizam GSTT1 i GSTM1 gena u limfocitima periferne krvi pacijentkinja u zavisnosti od stadijuma intracervikalne lezijesr
dc.titleMicronucleus frequency and polymorphism of GSTT1 and GSTM1 genes in peripheral blood lymphocytes of patients related to the stage of intracervical lesionen
dc.typedoctoralThesisen
dc.rights.licenseBY-NC-ND
dcterms.abstractМилошевић-Ђорђевић, Оливера; Јоксић, Гордана; Aрсенијевић, Слободан; Грујичић, Дарко; Стошић, Ивана; Фреквенца микронуклеуса и полиморфизам ГСТТ1 и ГСТМ1 гена у лимфоцитима периферне крви пацијенткиња у зависности од стадијума интрацервикалне лезије; Фреквенца микронуклеуса и полиморфизам ГСТТ1 и ГСТМ1 гена у лимфоцитима периферне крви пацијенткиња у зависности од стадијума интрацервикалне лезије;
dc.identifier.fulltexthttps://nardus.mpn.gov.rs/bitstream/id/47421/Disertacija.pdf
dc.identifier.fulltexthttps://nardus.mpn.gov.rs/bitstream/id/47422/ivana_stosic_23062015.pdf
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/47421/Disertacija.pdf
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/47422/ivana_stosic_23062015.pdf
dc.identifier.rcubhttps://hdl.handle.net/21.15107/rcub_nardus_3631


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