Ispitivanje prediktivnog i dijagnostičkog značaja pojedinačnih i kombinovanih biomarkera lipidnog statusa i inflamacije kod pacijenata sa kolorektalnim karcinomom
Investigation of predictive and diagnostic significance of single and combined biomarkers of lipid status and inflammation in patients with colorectal cancer
Докторанд
Mihajlović, MarijaМентор
Zeljković, AleksandraЧланови комисије
Spasojević-Kalimanovska, VesnaStefanović, Aleksandra
Ninić, Ana
Jovanović, Milan
Метаподаци
Приказ свих података о дисертацијиСажетак
Nedostatak pouzdanih serumskih biomarkera je jedan od osnovnih problema u dijagnostici i
proceni rizika za razvoj kolorektalnog karcinoma (colorectal cancer, CRC). Imajući u vidu da se razvoj
malignih bolesti odlikuje kako promenama ćelijskog metabolizma, tako i izmenama imunog,
oksidativnog i inflamatornog okruženja malignih ćelija, integrativni pristup, koji bi obuhvatio
istovremeno određivanje više biomarkera tipičnih za navedena stanja, predstavlja obećavajuću strategiju
u proceni rizika, skriningu i ranoj dijagnostici CRC.
U ovoj studiji je ispitivan potencijal pojedinačnih i kombinovanih biomarkera za dijagnostiku i
predviđanje rizika za nastanak CRC. Istraživanje je obuhvatilo četiri aspekta preko kojih se stekao uvid
u promene inflamatornog statusa, energetskog metabolizma i imune homeostaze pacijenata sa CRC. U
te svrhe smo određivali plazmatske koncentracije insulinu sličnog faktora rasta 1 (IGF1) i holesterola
sadržanog u lipoproteinima visoke gustine (HDL-h) kao indikatora ...metaboličkih promena, te rezistina i
E-kadherina kao parametara inflamacije. Drugi aspekt je obuhvatio ispitivanje funkcionalnosti enzima
uključenih u remodelovanje (lecitin-holesterol aciltransferaze, LCAT, holesterol-estar transfernog
proteina, CETP) i antioksidativnu funkciju (paraoksonaze 1, PON-1) HDL čestica kao zajedničkog
imenitelja energetskog metabolizma, redoks, imune i inflamatorne ravnoteže. Treći aspekt je činilo
određivanje nivoa ekspresije gena uključenih u kontrolu inflamatornog procesa (gena rezistina i njegovog
receptora, proteina udruženog sa adenilat ciklazom 1, CAP-1), imunog odgovora (gena faktora nekroze
tumora α, TNF-α) i metaboličke homeostaze (gena adiponektinskih receptora, Adipor1 i Adipor2) u
mononuklearnim ćelijama periferne krvi (PBMC). Konačno, četvrti aspekt predstavljala je procena
genetske predispozicije pojedinca za razvoj karcinoma bazirana na ispitivanju prisustva polimorfizama
pojedinačnih nukleotida gena od interesa (IG1 rs2946834, ADIPOQ rs266729, ADIPOR1 rs7539542,
ADIPOR1 rs1342387, TNF-α rs1800629, RETN rs1862513). Istraživanje je dopunjeno bioinformatičkom
analizom online dostupnih transkriptomskih baza podataka.
U istraživanje je uključeno 126 pacijenata sa CRC sa Klinike za opštu hirurgiju Vojnomedicinske
akademije u Beogradu, kao i 101 zdrava osoba koja je pristupila redovnom godišnjem lekarskom
pregledu u Opštoj bolnici Medigroup u Beogradu. Ispitanici su anketirani, te su na taj način prikupljeni
podaci o uzrastu, osnovnim antropometrijskim merama i životnim navikama. Rutinski biohemijski
parametri su određeni na analizatoru ILAB 300+, korišćenjem standardnih enzimskih i kolorimetrijskih
procedura. Aktivnosti LCAT i CETP su procenjene merenjem brzine stvaranja i prenosa holesterol estara.
Paraoksonazna aktivnost PON-1 je određena spektrofotometrijski uz paraokson kao supstrat, dok je
arilesterazna aktivnost određena metodom sa fenilacetatom. Lančana reakcija polimerizacije
(polymerase chain reaction, PCR) je korišćena u svrhe genotipizacije ispitanika i procene nivoa
ekspresije gena. Komercijalni ELISA testovi su korišćeni za određivanje plazmatskih koncentracija
IGF1, E-kadherina, rezistina, kao i CETP. ..
The lack of reliable serum biomarkers is among major problems linked to the diagnosis of
colorectal cancer (CRC) and the risk assessment for the disease development. Given that the development
of malignant diseases is characterized by changes in cellular metabolism and immune, oxidative and
inflammatory environment of malignant cells, an integrative approach, which includes simultaneous
determination of multiple biomarkers, represents a promising strategy for the risk evaluation, screening
and early diagnosis of CRC.
In this study, capacities of single and combined biomarkers for diagnosing and predicting the risk
of developing CRC were examined. The research included four approaches for the assesment of
alterations of inflammatory status, energy metabolism and immune homeostasis in CRC patients. For
these purposes, plasma concentrations of insulin-like growth factor 1 (IGF1) and high-density lipoprotein
cholesterol (HDL-c) as indicators of metabolic changes were determined, while resi...stin and E-cadherin
concentrations were used as indicators of inflammatory status. The second approach included testing the
functionality of enzymes involved in remodeling (lecithin cholesterol acyltransferase, LCAT, cholesterol
ester transfer protein, CETP) and antioxidative function (paraoxonase 1, PON-1) of HDL particle, which
was appreciated as a common denominator of energy metabolism, redox, immune and inflammatory
homeostasis. The third approach was to determine the levels of gene expressions involved in the control
of the inflammatory processes (resistin and its receptor, adenylate cyclase-associated protein 1, CAP-1),
immune response (tumor necrosis factor α, TNF-α) and metabolic homeostasis (adiponectin receptors,
Adipor1 and Adipor2) in peripheral blood mononuclear cells (PBMCs). Finally, the fourth approach was
the assessment of an individual's genetic predisposition to develop CRC, which was based on the
exploration of specific single nucleotide polymorphisms (SNPs): IGF1 rs2946834, ADIPOQ rs266729,
ADIPOR1 rs7539542, ADIPOR1 rs1342387, TNF-α rs1800629, RETN rs1862513. The research was
further supplemented by a bioinformatics analysis of online available transcriptome databases.
The study included 126 patients with CRC from the Clinic for General Surgery of the Military
Medical Academy in Belgrade, as well as 101 healthy individuals who attended the regular annual
medical examination at the General Hospital Medigroup in Belgrade. The participants were interviewed,
and data on age, basic anthropometric measurments and life habits were collected. Routine biochemical
parameters were determined on ILAB 300+ analyzer, using standard enzymatic and colorimetric
procedures. LCAT and CETP activities were assessed by measuring the rate of formation and transfer of
cholesteryl esters. Paraoxonase activity of PON-1 was determined spectrophotometrically with
paraoxone as a substrate, while arylesterase activity was determined by the phenylacetate method.
Polymerase chain reaction (PCR) was used for genotyping and the assessment of gene expression levels.
Commercial ELISA assays were used to determine plasma concentrations of IGF1, E-cadherin, resistin,
and CETP. ..