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Mikrosatelitska nestabilnost i gubewe na heterozigotnost kaj
pacienti so kolorektalen karcinom od Republika Makedonija
Hiqadnikova-Bajro M.
1
, Josifovski T.
2
, Sterjev Z.
1
, Kapedanovska A.
1
, Serafimoska Z.
1
, Matevska N.
1
,
Despotovska S.
1
, Petru{evska N.
3
, Panovski M.
2
, [uturkova Q.
1
, Dimovski A. J.
1
1
Institut za farmacevtska hemija, Farmacevtski fakultet, 
2
Klinika za Abdominalna hirurgija i
3
Institut za Radioterapija i Onkologija, Medicinski Fakultet, 
Univerzitet ”Sv. Kiril i Metodij”, Skopje, Republika Makedonija
Genomskata nestabilnost se smeta za klu~en molekularen i patogenetski ~ekor vo ranata tumoroge-
neza, koj sozdava povolna sredina za pojava na promeni vo tumor supresornite geni i onkogenite. Kaj kolo-
rektalniot karcinom (KRK) postojat najmalku tri formi na genetska nestabilnost: mikrosatelitska nes-
tabilnost (Microsatellite instability MSI), hromozomska nestabilnost t.e. aneuzomija, duplikacii ili delcii
na delovi od hromozomite (chromosomal instabillity CIN) i hromozomski translokacii. Rasvetluvaweto na
pri~inata i specifi~nata uloga na genomskata nestabilnost vo KRK stanuvaat neophodni za vospostavuva-
we poefikasni dijagnosti~ki, prognosti~ki i hemoterapevtski strategii. Ovaa studija se fokusira kon
definirawe na genomskata nestabilnost odnosno MSI i gubitokot na heterozigotnost (loss of heterozygos-
ity-LOH) kaj 238 slu~ajno odbrani pacienti so KRK od Republika Makedonija koi bile podlo`eni na kolek-
tomija pri Klinikata za Abdominalna hirurgija vo Skopje. Genomska DNK e izolirana od periferna krv
i sve`o tkivo vedna{ po izvr{enata operacija so primena na Proteinaza K digestija, fenol/hloroform
ekstrakcija i etanol precipitacija. MSI i LOH statusot vo tumorot be{e odreduvana so fluorescentna
multipleks polimeraza veri`na reakcija i posledovatelna kapilarna gel elektroforeza na Genetski
Analizator AbiPrism310. Evaluacijata vklu~uva{e 14 mikrosatelitski markeri locirani na 1p, 2p, 4q, 5q,
8p, 17p, 17q, 18q, odbrani spored preporakite od NCI odnosno locirani vo regioni koi sodr`at tumor supre-
sorni geni ili onkogeni (APC, p53, LMYC, EphB2 MSH2 cKIT, SMAD4). MSI be{e najdena kaj 22/238 (9.2%) od
karcinomite, i toa so po~esta zastapenost kaj tumori locirani vo proksimalniot kolon i tumori so ponizok
TNM stadium. Pove}e od polovina od pacientite (116/208) manifestiraat LOH na barem eden hromozomski
krak, pri {to 72% od niv poka`aa disbalans na nivo na pove}e markeri (CIN-high status). Najvisok stepen na
LOH be{e detektiran kaj hromozomot 18q (50%), a po nego sleduvaat 1p(35%), 5q(34%),17p(28%), 17q(26%),
8p(24%) i 2p(18%). D18S61 e marker so najvisoka nivo na LOH kaj pacientite so KRK od R. Makedonija. Kaj
edna tretina od site slu~ai ne e detektiran MSI i LOH vo tumorskoto tkivo, {to uka`uva na postoewe na
drug mehanizam vo etiopatogenezata na kolorektalniot karcinom kaj pacientite od na{ata dr`ava. 
Macedonian pharmaceutical bulletin 53 (1,2) 158-159 (2007)
PP - 72
159
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IGF1 gene promotor variant as risk modifier for colorectal cancer
M. Hiljadnikova-Bajro
1
, T. Josifovski
2
, Z. Sterjev
1
, A. Kapedanovska
1
, Z. Serafimoska
1
,
N. Matevska
1
, S. Despotovska
1
, N. Petrusevska
3
M. Panovski
2
, L. Suturkova
1
, A. J. Dimovski
1
1
Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, 
2
Clinic for Abdominal Surgery 
and 
3
Institute of Radioterapy and Oncology, Faculty of Medicine, 
University “Sts. Cyril and Methodius“, Skopje, Republic of Macedonia
Insulin-like growth factor I (IGF1)is proved to be involved in colorectal cancerogenesis, with implication on
cell transformation, tumor growth, metastasis and poor prognosis. The length of the CA repeat element in the promot-
er region of IGF1 is associated with transcription and plasma levels of IFG1. The aim of the study was to investigate
the role of IGF1 length polymorphisms in risk of colorectal cancer in a case control study of 102 colorectal cancer
patients and 71 controls. The CA repeat numbers in IGF1 promoter were determined by fluorescent PCR and capillary
gel electrophoresis. Nine different alleles were found among CRC patients with the following frequency: CA
12
(0.5%),
CA
16
(0.5%), CA
17
(1.5%) CA
18
(5.9%) CA
19
(66.6%) CA
20
(18.1%) CA
21
(4.9%), CA
22
(1.5%), CA
23 
(0.5%). The cor-
responding frequencies among healthy controls were as follows: CA
11
(0.8%), CA
17
(0.7%), CA
18
(8.4%) CA
19
(65.5%)
CA
20
(18.3%) CA
21
(4.9%), CA
22
(1.4%). Similar allele frequencies and genotype distributions were identified for
the most common CA
19
allele, as well as for the CA
<18
and CA
>19
alleles in patients and controls. However, signif-
icantly higher frequency of CA
<18
allele was found in CRC patients <50 years of age compared both to patients >50
years of age and to normal controls, indicating that shorter CA repeat in the IGF1 gene promoter might act a genet-
ic risk factor for early development of CRC in our population. Further studies will be conducted to determine whether
this polymorphism is a risk factor by itself or, additional factors that influence IGF1 plasma levels like physical activ-
ity, diet and smoking accelerate cancerogenesis in younger population.
Macedonian pharmaceutical bulletin 53 (1,2) 160-161 (2007)
PP - 73
160
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Varijanti vo promoterot na IGF1 genot vlijaat na rizikot 
za razvoj na kolorektalen karcinom
Hiqadnikova-Bajro M.
1
, Josifovski T.
2
, Sterjev Z.
1
, Kapedanovska A.
1
, Serafimoska Z.
1
, Matevska
N.
1
, Despotovska S.
1
, Petru{evska N.
3
, Panovski M.
2
, [uturkova Q.
1
, Dimovski A. J.
1
1
Institut za farmacevtska hemija, Farmacevtski fakultet, 
2
Klinika za Abdominalna hirurgija 

3
Institut za Radioterapija i Onkologija, Medicinski Fakultet, 
Univerzitet ”Sv. Kiril i Metodij”, Skopje, Republika Makedonija
Doka`ano e deka Insulin-sli~niot faktor za rast 1 (IGF1) igra gloga vo kolorektalnata kancero-
geneza so involviranost vo kleto~nata transformacija, tumorskiot rast i sposobnosta za metastazirawe
na tumorite. Dol`inata na SA povtoruva~kiot element vo promoterskiot region na IGF1 e asocirana so
traskripcijata i nivoata na IGF1 vo plazmata. Cel na ovaa studija e da se ispita zna~eweto na polimor-
fizmot vo dol`inata na IGF1 za rizikot od pojava na kolorektalen karcinom preku studija koja vklu~uva
102 pacienti so kolorektalen karcinom (KRK) i kontrolna grupa od 71 zdravi individui. Brojot na CA
povtoruvawa vo IGF1 promoterot be{e opredelen so fluorescentna polimeraza veri`na reakcija i kapi-
larna gel elektroforeza. Kaj pacientite bea identifikuvani devet razli~ni aleli so slednata frekven-
cija CA
12
(0.5%), CA
16
(0.5%), CA
17
(1.5%) CA
18
(5.9%) CA
19
(66.6%) CA
20
(18.1%) CA
21
(4.9%), CA
22
(1.5%), CA
23
(0.5%).
Kaj kontrolnata grupa bea najdeni vkupno 9 razli~ni alelei so slednata frekvencija: CA
11
(0.8%),
CA
17
(0.7%), CA
18
(8.4%) CA
19
(65.5%) CA
20
(18.3%) CA
21
(4.9%), CA
22
(1.4%). Sli~na alelna frekvencija i genotip-
ska distribucija za naj~estiot CA
19
alel kako i za CA<
18
i CA<
19
alelite bea utvrdeni kaj pacientite i
kontrolite. Koga pacientite bea podeleni spored razli~ni klini~ki i patohistolo{ki parametri, sig-
nifikantno povisoka frekvencija na CA<
18
alelot be{e zabele`ana kaj pacienti so KRK na vozrast pod
pedeset godini, vo sporedba so pacientite postari od 50 godini kako i vo sporedba so kontrolnata grupa.
Ovie rezultati uka`uvaat deka pokratkiot SA povtoruva~ki element vo promoterot na IGF1 genot mo`e
da se odnesuva kako genetski rizik faktor za rano razvivawe na kolorektalniot karcinom vo na{ata pop-
ulacija. Ponatamo{ni studii }e bidat sprovedeni za da se utvrdi dali ovoj polimorfizam pretstavuva
rizik faktor sam po sebe ili vo sodejstvo so faktori na sredinata koi vlijaat vrz plazma koncentraci-
ite na IGF1, kako fizi~kata aktivnost, ishranata i pu{eweto.
Macedonian pharmaceutical bulletin 53 (1,2) 160-161 (2007)
PP - 73
161
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Genetic Polymorphism of Manganese Superoxide Dismutase
(MnSOD) and Prostate Cancer Susceptibility
Arsova-Sarafinovska Z., Matevska N., Despotovska S., Petrovski D., Dzikova S., 
Banev S., Georgiev V., Sikole Ad, Dimovski A. J
Department for Drug Quality Control, Republic Institute for Health Protection, 
Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, Clinic of Urology, Department of Nephrology, 
Institute for Pathology, Faculty of Medicine, Skopje, Macedonia.
Prostate cancer continues to be the most frequently diagnosed neoplasm, and the second leading cause of
cancer-related mortality in men. Oxidative stress may enhance prostatic carcinogenesis. MnSOD, the only known
superoxide scavenger in mitochondria, may be particularly important for antioxidant defence because mitochondria
are major cites for cellular metabolism and, hence production of reactive oxygone species (ROS). A T C single
nucleotide substitution, resulting in a Val Ala change at the -9 position (Val-9Ala), which alters the secondary struc-
ture of the protein, has been noted to affect transport of MnSOD into the mitochondria. The Ala allele was associat-
ed with an increased risk of breast cancer, prostate cancer and of early-onset colorectal cancer while the Val allele
was associated with an increased risk of lung and bladder cancer. 
We have determined MnSOD genotype in 60 prostate cancer cases and 167 BPH patients. Val-9Ala polymor-
phism in the signal sequence of the protein for MnSOD was determined using the real time polymerase chain reac-
tion (PCR) amplification with using TaqMan fluorescently labelled probes. We have found no significant difference
in prostate cancer susceptibility in the subjects with Ala/Ala genotype as compared with those with Val/Val and Val/Ala
genotype (OR: 0.9318; 95% CI: 0.4729 - 1.8361, p= 0.8378). We did not observe an association of the Ala/Ala geno-
type and the age of the prostate cancer patients (OR: 1.8824; 95% CI: 0.5785 - 6.1253, p= 0.2923). However, the fre-
quency of Ala/Ala genotype was slightly elevated in the patients with higher tumour grade as compared to patient with
lower tumor grade (Gleason score (8-10) vs. Gleason score (2-6); OR: 3.75; 95% CI: 1.0476-13.4236, p= 0.0320).
This data suggest that Ala/Ala MnSOD genotype could contribute in the increased risk of progression of
prostate cancer in patients from Republic of Macedonia.
Macedonian pharmaceutical bulletin 53 (1,2) 162-163 (2007)
PP - 74
162
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Val9Ala MnSOD varijantata vlijae na progresijata 
na karcinomot na prostatata kaj pacienti 
od Republika Makedonija
Arsova-Sarafinoska Z., Matevska. N., Despotovska S., Petrovski D., Xikova S.,
Banev. S, Georgiev. V, [ikole A., Dimovski A. J.
Institut za kontrola na kvalitet na lekovi, Republi~ki zavod za zdravstvena za{tita, 
Institut za Farmacevtska hemija, Farmacevtski Fakultet, Klinika za uroligija, 
Institut za nefrologija, Institut za patologija, Medicinski Fakultete, Skopje, Makedonija
Karcinomot na prostata prodol`uva da bide naj~esto dijagnosticirana neoplazma i vtora vode~ka
pri~ina za kancer-asocirana smrtnost kaj ma{kata populacija. Oksidativniot stres mo`e da go pottikne
procesot na karcinogeneza vo prostatata. MnSOD, edninstveniot poznat neutrolizator na superoksidnite
joni vo mitohondriite, ima delumno vlijanie vo procesot na antioksidativna za{tita. Zabele`ano e deka
T C nukleotidnata supstitucija, koja rezultitira so Val Ala promena na 9-tata pozicija (Val-9Ala) i prome-
na vo sekundarnata struktura na proteinot, vlijae na transportot na MnSOD vo vnatre{nosta na mitohon-
driite. Ala alelot e asociran so zgolemen rizik za razvoj na kancer na dojka, kancer na prostata i rana poja-
va na kolorektalen kancer, dodeka Val alelot e asociran so zgolemen rizik za razvoj na kancer na beli
drobovi i kancer na mo~en meur. 
Nie go odredivme MnSOD genotipot kaj 60 pacienti so kancer na prostata i 167 BPH pacienti. Val-
9Ala
polimorfizmot vo signalnata sekvenca na proteinot be{e odredena so primena na metodata na real-time
PCR i fluorescentno odbele`ani TaqMan probi. Statisti~kata obratotka na dobienite rezultati poka`a
deka ne postoi zna~ajna razlika vo genotipskata frevencija na Ala/Ala, Val/Val Val/Ala genotipovite pome|u
pacientite i kontrolite (OR: 0.9318; 95% CI: 0.4729 - 1.8361, p= 0.8378). Isto taka ne zabele`avme asoci-
jacija na Ala/Ala genotipot i vozrasta na pacientite so kancer na prostata (OR: 1.8824; 95% CI: 0.5785 -
6.1253, p = 0.2923). Me|utoa, zabele`avme deka frekfencijata na Ala/Ala genotipot e zgolemena kaj pacien-
tite kaj koi pri dijagnoza tumorot e vo ponapreden stadium (Gleason score 8-10) sporedeni so pacientite
kaj koi tumor e dijagnosticiran vo poran stadium na bolesta (Gleason score 2-6); (OR: 3.75; 95% CI: 1.0476-
13.4236, p= 0.032).
Ovie podatoci uka`uvaat deka Ala/Ala MnSOD genotipot pridonesuva za zgolemen rizik za progre-
sija na karcinomot na prostata kaj pacientite od Republika Makedonija.
Macedonian pharmaceutical bulletin 53 (1,2) 162-163 (2007)
PP - 74
163
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Proteomics of Synechocystis sp.PCC6803
Towards identification of plasma membrane protein complexes
Aleksandra Kapedanovska
1,2,
Birgitta Norling
2
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Skopje, R.Macedonia
2
Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
Cyanobaceria constitute the largest group of photosynthetic prokaryotes because of their wide spread
occurrence, frequent abundance and morphological diversity. Synechocystis sp.PCC6803 is unicellular, naturally
transformable cyanobacetria and is particularly attractive model system for genetic and biochemical studies of pho-
tosynthesis and other metabolic processes. Recently, BN-PAGE combined with 2
nd
dimension SDS-PAGE has been
used to detect specific interactions between large protein complexes that has led to the discovery of so-called 'super
complexes', which are not anymore only  an exception but it is more clearly that they are becoming basic working
principle of the cell. This method has already been used to analyze photosynthetic complexes of thylacoid membranes
of Synechocystis, but not the plasma membrane complexes. 
In the present work we are trying to establish a proteomic approach suitable to identify the composition and
dynamics of plasma membrane protein complexes of cianobacterium Synechocystis sp.PCC6803 using two dimen-
sional Blue native/ SDS PAGE. We have however found that applying the same conditions as described in previ-
ous studies (1) do not resolve any complexes in the plasma membrane of Synechocystis, although there were results
on the thylacoid membranes. This may be dye to a different lipid composition of the two membranes. Finding suit-
able detergents for the solubilization of different protein complexes is a key for a wider application of BN-PAGE
in the investigation of membrane protein complexes. A suitable detergent must be as mild as possible but able to
disrupt lipid-lipid interactions without disturbing those between protein components in complexes and should also
not interfere with the electrophoresis process. Comparative testing of different detergents in various detergent-to-pro-
tein ratios in the presence of different salts is necessary to determine the optimal detergent and the right conditions
for solubilization. 
We have preliminary results that show the presence of a number of complexes in the plasma membrane.
1.Mirkka Herranen, Natalia Battchikova, Pengpeng Zhang, Alexander Graf, Sari Sirpio, Virpi Paakkarinen,
and Eva-Mari Aro, (January 2004)  Plant Physiology, 134 , 470-481
Macedonian pharmaceutical bulletin 53 (1,2) 164-165 (2007)
PP - 75
164
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Proteomika na Synechocystis sp. PCC6803
~ekor napred vo identifikacijata 
na proteinskite plazma mebranski kompleksi
Aleksandra Kapedanovska
1,2,
Birgitta Norling
2
1
Institut po farmacevtska hemija , Farmacevtski fakultet, Skopje, R.Makedonija 
2
Institut po biohemija i biofizika , Stoholm Univerzitet; Stoholm;  [vedska
Cianobakteriite ja so~inuvaat najgolemata grupa na fotosintetski prokarioti karakteristi~-
ni zaradi nivnata golema zastapenost, {iroka rasprostranetost i morfolo{ki diverzitet. Unicelular-
nata cianobakteria Synechocystis sp.PCC6803 prestavuva atraktiven sistemski model za niza genetski i
biohemiski studii na fotosintetskite i metabolni procesi na cianobakteriite poradi nejzinata lesna
kultivacija, prirodno transformabilnost i celosno poznat genom. Vo ponovo vreme, metodata koja gi kom-
binira BN-PAGE vo prva dimenzija i SDS-PAGE vo vtora dimenzija se koristi za detekcija i analiza na
spesifi~ni interakcii pome|u golemi proteinski kompleksi {to vodi do otkrivawe na t.n super kom-
pleksi, koi pove}e ne se isklu~ok tuku nesomneno e deka prestavuvaat osnoven princip na funkcionira-
we na kletkata. Ovaa elektroforetska tehnika se poka`ala kako uspe{na vo prou~uvaweto na fotosintet-
skite kompleksi na tilakoidnite membrani na Synechocystis, no seu{te nema nau~ni soznanija za postoewe
na plazma membranski kompleksi.
Celta na ovoj trud e da se pronajde proteomi~en pristap pogoden za identifikacija na sostavot
i dinamikata na proteinskite plazma membranski kompleksi na Synechocystis sp.PCC6803 preku upotreba
na dvodimenzionalna Blue native/ SDS PAGE. So dosega{nata rabota dojdovme do zaklu~ok deka uslovite
koristeni vo predhodnite studii (1) ne se pogodni za razdeluvawe na kompleskite vo plazma membranite
na Synechocystis, iako istite se poka`ale kako pogodni vo razdeluvaweto na proteinskite kompleksi vo
tilakoidnite membrani. Predpostavuvame deka razli~niot lipiden sostav pome|u plazma membranite i
tilakoidnite membrani e pri~ina za vakvite rezultati. Izborot na soodveten detergent potreben za sol-
ubilizacija na razli~nite proteinski kompleksi prestavuva klu~en ~ekor vo uspe{na BN-PAGE. Deter-
gentot treba da ispolnuva nekolku uslovi me|u koi najbitno e da bide neinvaziven, no istovremeno i
dovolno "jak" za da gi razori lipidno-lipidnite vrski bez pritoa da deluva na vrskite pome|u protein-
skite subedinici vo kompleksite kako i da bide indiferenten vo odnos na elektroforezata. Neophodni
se komparativni testirawa, koi vklu~uvaat nekolku razli~ni detergenti vo uslovi na razli~ni protein
- detergent odnosi, so cel da se odbere najsoodvetniot detergent i najsoodvetnite uslovi za solubiliza-
cija. So dosega{nata rabota dobivme preliminarni rezultati koi uka`uvaat na prisustvoto na golem broj
na proteinski kompleksi vo plazma membranite.
1.Mirkka Herranen, Natalia Battchikova, Pengpeng Zhang, Alexander Graf, Sari Sirpio, Virpi Paakkarinen, and
Eva-Mari Aro, (January 2004) Plant Physiology, 134, 470-481
Macedonian pharmaceutical bulletin 53 (1,2) 164-165 (2007)
PP - 75
165
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Prostate Cancer Risk 
and Glutathione Peroxidase 1 Codon 198 Variant
Arsova-Sarafinovska Z., Matevska N., Despotovska S., Petrovski D., Dzikova S, 
Banev S., Georgiev V., Sikole A., Dimovski A. J.
Department for Drug Quality Control, Republic Institute for Health Protection, 
Department of Molecular Biology and Genetics, Institute of Pharmaceutical Chemistry, Faculty of Pharmacy, 
Clinic of Urology, Department of Nephrology, Institute for Pathology, Faculty of Medicine, Skopje, Macedonia.
An increased oxidative stress, a shift in the prooxidant-antioxidant balance toward a more oxidative state,
is known to cause DNA damage and mutations of cancer promoting tumor suppressor genes, initial events in carcino-
genesis. Glutathione peroxidase GPX1 is ubiquitously expressed selenium-dependent enzyme that protects cells
against oxidative damage by reducing hydrogen peroxide and a wide range of organic peroxides with reduced glu-
tathione. It was reported that a C T substitution at codon 198 of the GPX1 gene which results in a proline (Pro) to
leucine (Leu) substitution and the GPX1 variant was significantly associated with an increased risk of lung, breast
and bladder cancer risk. 
We have tested GPX1 genotype in 60 prostate cancer cases and 167 benign prostatic hyperplasia (BPH) patients
using the real time polymerase chain reaction (PCR) amplification using TaqMan fluorescently labelled probes.
The frequencies of Pro/ProPro/Leu and Leu/Leu genotypes were found to be: 0.57, 0.28, 0.15 and 0.56,
0.32, 0.12 in prostate cancer cases and BPH cases, respectively. There was no significant difference in GPX1 geno-
type frequency between cancer and BPH group (OR: 1.2971; 95% CI: 0.5551- 3.0309; p= 0.5530). We did not observe
an association of the Pro/Pro genotype and the age of the prostate cancer patients (OR: 1.1429; 95% CI: 0.274-4.767;
p=0.8548). Additionally, there was no difference in GPX1 genotype frequency in the patients with high and low
tumor grade (Gleason score (8-10) vs. Gleason score (2-6), OR: 0.9; 95% CI: 0.2164-3.7427, p= p= 0.8846).
The results of this study suggest that the GPX1 genotype is unlikely to be associated with the risk of devel-
oping prostate cancer in patients from the Republic of Macedonia.
Macedonian pharmaceutical bulletin 53 (1,2) 166-167 (2007)
PP- 76
166
^ETVRTI KONGRES NA FARMACIJATA NA MAKEDONIJA SO ME\UNARODNO U^ESTVO
FOURTH CONGRESS OF PHARMACY OF MACEDONIA WITH INTERNATIONAL PARTICIPATION

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