Title Obilježja stanične imunosti u bolesnika s benignom prostatičnom hiperplazijom i karcinomom prostate : doktorski rad
Title (english) Characteristics of cellular immunity in patients with benign prostatic hyperplasia and Prostatic Cancer
Author Stanislav Sotošek
Mentor Ines Mrakovčić-Šutić (mentor)
Committee member Anton Maričić (predsjednik povjerenstva)
Committee member Sunčana Kukolja-Taradi (član povjerenstva)
Committee member Bojan Polić (član povjerenstva)
Committee member Ines Mrakovčić-Šutić (član povjerenstva)
Granter University of Rijeka Faculty of Medicine Rijeka
Defense date and country 2011-01-01, Croatia
Scientific / art field, discipline and subdiscipline BIOMEDICINE AND HEALTHCARE Clinical Medical Sciences Urology
Universal decimal classification (UDC ) 616 - Pathology. Clinical medicine
Abstract Cilj istraživanja: Benigna prostatična hiperplazija (BPH) i karcinom prostate (CaP), nakon
infekcija mokraćnog sustava, dva su najčešća stanja s kojima se urolog susreće u svojoj
svakodnevnoj praksi. Oba klinička entiteta predstavljaju "bolesti starije muške populacije", te
njihova učestalost raste s godinama. Tijekom zadnjih nekoliko godina, istraživanja su ukazala
na moguću ulogu imunološkog sustava u patogenezi BPH i CaP, no točan mehanizam
njihovog razvoja i progresije još uvije nije razjašnjen. Cilj ovog rada bio je istražiti promjene
stanične imunosti, poglavito udjela leukocitnih subpopulacija, izražavanje citotoksičnog
posrednika perforina, aktivnost stanica NK i izražaj pojedinih citokina u različitim
imunokompetentnim stanicama periferne krvi i tkivu prostate bolesnika s BPH i CaP.
Ispitanici, materijal i metode: U istraživanje je bilo uključeno 40 bolesnika s BPH i 40
bolesnika s CaP. Zdravi, dobrovoljni davatelji krvi bili su kontrolna skupina. Demografski,
klinički i laboratorijski podatci (vrijednosti prostata specifičnog antigena) prikupljani su i
analizirani za svakog bolesnika. Svakom bolesniku s BPH i CaP uzimali smo uzorak periferne
krvi i tkiva prostate. Te smo uzorke slali na imunološka istraživanja. Mononuklearne stanice
tkiva prostate izdvojili smo enzimatskom digestijom s 0,1% kolagenazom i naslojavanjem na
gradijentu gustoće. Mononuklearne stanice periferne krvi također smo izdvojili
centrifugiranjem na gradijentu gustoće. Višestrukim obilježavanjem površinskih biljega
metodom izravne imunofluorescencije analizirali smo fentotip mononuklearnih stanica
periferne krvi i tkiva prostate. Izdvojene mononuklearne stanice periferne krvi i tkiva prostate
obilježavali smo istovremeno protutijelima na površinske biljege kako bi utvrdili njihovu
pripadnost pojedinim leukocitnim populacijama, permeabilizirali njihovu membranu te
metodom izravne i neizravne imunofluorescencije unutarstanično obilježavali perforin,
interleukin IL-4 i IFNγ (interferon gama) korištenjem odgovarajućih monoklonskih
protutijela, te smo stanice analizirali na protočnom citometru. Uzorci koji su bili predviđeni
za unutarstanično obilježavanje citokina prethodno smo stimulirali s PMA (engl. phorbol
myristate acetat), ionomicinom i monenzinom radi prikazivanja citokina u stanici.
Imunofluorescenciju smo koristili za prikazivanje molekule perforina u preparatima tkiva
prostate. Metodom pozitivne magnetske separacije iz mononuklearnih stanica periferne krvi
izdvojili smo stanice NK, te smo izdvojene stanice NK i mononuklearne stanice tkiva prostate
stavili u dvosatni test citotoksičnosti prema K-562 NK osjetljivoj staničnoj liniji uz očitavanje
protočnim citometrom.
Rezultati: Bolesnici s CaP su bili značajno stariji i imali su statistički značajno veće
vrijednosti PSA od bolesnika s BPH. Udio limfocita T te njegovih CD3+CD4+CD56- i
CD3+CD8+CD56- subpopulacija nije se statistički značajno razlikovao u perifernoj krvi niti u
tkivu prostate bolesnika s BPH i bolesnika s CaP. Distribucija CD3+ stanica uočena je
uglavnom u epitelu kontrolnog tkiva prostate te tkiva prostate bolesnika s BPH i bolesnika s
CaP. Učestalost CD4+CD25+FoxP3+ stanica u perifernoj krvi i tkivu prostate bila je statistički
značajno veća u bolesnika s CaP. Iako se udio stanica NK u perifernoj krvi bolesnika s BPH,
bolesnika s CaP te zdravih dobrovoljnih davatelja krvi nije statistički značajno razlikovao,
udio stanica NK te njihove CD3-CD56+dim subpopulacije je bio statistički značajno niži u
tkivu prostate bolesnika s CaP. Distribucija CD56+ stanica uočena je u stromi tkiva prostate
bolesnika s BPH, bolesnika s CaP te u kontrolnome tkivu prostate. Izražena hiperplazija i
povećana infiltracijom CD56+ stanica uočena je u stromi tkiva prostate bolesnika s BPH. Udio
stanica NKT bio je statistički značajno viši u perifernoj krvi bolesnika s BPH u odnosu udio
stanica NKT u perifernoj krvi bolesnika s CaP i u kontrolnoj skupini. Udio stanica NKT
izoliranih iz tkiva prostate bolesnika s BPH i bolesnika s CaP nije se statistički značajno
razlikovao. Distribucija stanica NKT uočena je u epitelu kontrolnoga tkiva prostate znatno
više negoli u epitelu tkiva prostate bolesnika s BPH i bolesnika s CaP. Udio limfocita B bio je
statistički značajno viši u perifernoj krvi bolesnika s BPH u odnosu udio limfocita B u
perifernoj krvi bolesnika s CaP i u kontrolnoj skupini. Udio limfocita B izoliranih iz tkiva
prostate bolesnika s BPH i bolesnika s CaP nije se statistički značajno razlikovao. Udio
perforin pozitivnih stanica bio je statistički manji u tkivu prostate bolesnika s CaP u odnosu
na bolesnike s BPH. Manji udio perforina uočen je u CD3+CD56- limfocitima T te njegovim
CD3+CD4+CD56- i CD3+CD8+CD56- subpopulacijama, u stanicama NK i njihovoj CD3-
CD56+dim subpopulaciji te stanicama NKT izoliranih iz tkiva prostate bolesnika s CaP u
odnosu na bolesnike s BPH. Stanice NK izolirane iz periferne krvi bolesnika s BPH i
bolesnika s CaP imale su statistički značajno manju citotoksičnu aktivnost prema K-562
osjetljivoj staničnoj liniji u odnosu na stanice NK iz periferne krvi zdravih dobrovoljnih
davatelja krvi. Mononuklearne stanice izolirane iz tkiva prostate bolesnika s BPH i bolesnika
s CaP imale su malu citotokičnu aktivnost prema K-562 osjetljivoj staničnoj liniji. Statistički
veći udio IFN+ stanica nađen je samo u stanicama NK izoliranih u bolesnika s BPH, dok se
udio IFN+ i IL-4+ stanica nije statistički značajno razlikovao u ostalim limfocitnih
subpopulacijama izoliranih iz tkiva prostate bolesnika s BPH i CaP. Negativna korelacija
utvrđena je između postotaka CD3+CD56- stanica u perifernoj krvi i tkivu prostate te vrijednosti PSA u bolesnika s CaP, ali ne i u bolesnika s BPH. Pozitivnu korelaciju smo uočili
između postotka CD4+CD25+FoxP3+ stanica u perifernoj krvi i tkivu prostate te vrijednosti
PSA u bolesnika s CaP, ali ne i u bolesnika s BPH. Negativna korelacija nađena je između
perforin pozitivnih stanica, CD3+perforin+, CD56+perforin+ stanica te vrijednosti PSA samo u
tkivu prostate bolesnika CaP.
Zaključak: Rezultati ovog istraživanja jasno ukazuju da mali postotak stanica citotoksičnog
fenotipa, osobito stanica NK, mali udio perforin pozitivnih stanica, uključujući limfocite T,
stanice NK i stanice NKT, niska citotoksična aktivnost kao i povećanje udjela T regulacijskih
stanica u bolesnika s BPH, a osobito u bolesnika s CaP su posljedica lokalnih zbivanja u tkivu
prostate i jedan je od mogućih mehanizama u patogenezi i progresiji BPH i CaP. Potvrda ovih
rezultata zahtjeva daljnja istraživanja.
Abstract (english) Objectives: Benign prostatic hyperplasia (BPH) and prostatic carcinoma (CaP), following the
uroinfections, are the most common diseases in urologic everyday practice. Both entity
present “disease of aged male population” and their incidence rise with aging. Past several
years, the investigations showed possible role of immune system in the pathogenesis of BPH
and CaP, but precise mechanism of their development and progression is still unknown. The
purpose of the study was the changes in cell-mediated immunity, especially the frequency of
leukocytes subpopulations, expression of cytotoxic mediator perforin, activity of NK cells and
expression of cytokines in different immune cells in peripheral blood and prostatic tissue of
BPH and CaP patients.
Patients, material and methods: Fourthy patients with BPH and 35 patients with CaP were
included in the study. Healthy volunteers served as controls. Demographic, clinical and
laboratory data (values of prostate specific antigen) were collected and analyzed for each
patient. The samples of peripheral blood and prostatic tissue of PBH and CaP patients were
collected and sent further immunological investigations. Mononuclear cell for prostatic tissue
are separated by enzymatic digestion with 0.1% cllagenase and gradient centriguatlion.
Peripheral blood mononuclear cells were obtained after gradient centrifugation. The
phenotype of mononuclear cells for prostatic tissue and peripheral blood were analyzed by
simultaneous multiple staining of surface antigens using direct immunoflourescency and flow
cytometry analyzes. Isolated mononuclear cells were stained simultaneously by monoclonal
antibody against surface antigens, specific for different leukocyte populations, and were
further permeabilized for intracellular staining of perforin, interleukin IL-4 and IFNγ
(interferon gamma). The labeled cells were analyzed by flow cytometry. Cell samples that
were scheduled for intracellular cytokine staining were previously stimulated with PMA,
ionomycine and monensim which are necessary for cytokine visualization.
Immunofluorescency was used for visualization of perforin molecule in the samples of
prostatic tissue. CD56+ NK cells were isolated from peripheral blood mononuclear cells by
positive magnetic cell separation method. The cytotoxic activity against NK sensitive cell K-
562 cell line of separated CD56+ NK cells of peripheral blood and mononuclear cells isolated
form prostatic tissue were tested by 2 hours cytotoxic assay and was analyzed by flow
cytometry.
Results: Patients with CaP were significantly older and had significantly higher PSA levels
than patients with BPH. The frequency of T lymphocytes and its CD3+CD4+CD56- and CD3+CD8+CD56- subpopulations was not significantly different either in the peripheral blood
or in the prostatic tissue of patients with BPH and CaP patients. Distribution of CD3+ cells
was observed mainly in the epithelium of control prostate tissue and prostate tissue of patients
with BPH and CaP patients. The frequency of CD4+CD25+FOXP3+ cells in peripheral blood
and prostate tissue was significantly higher in patients with CaP. Although the frequency of
NK cells in peripheral blood of patients with BPH, patients with CaP and healthy volunteers
did not differ significantly, the frequency of NK cells and their CD3-CD56+dim subpopulation
was significantly lower in the prostatic tissue of patients with CaP. Distribution of CD56+
cells was observed in the stroma of prostatic tissue of patients with BPH, patients with CaP
and in control prostatic tissue. Marked hyperplasia and increased infiltration of CD56+ cells
was observed in the stroma of prostatic tissue of patients with BPH. The frequency of NKT
cells was significantly higher in the peripheral blood of patients with BPH than share NKT
cells in the peripheral blood of patients with CaP and the control group. The frequency of
NKT cells isolated from prostate tissue of patients with BPH and patients with CAP did not
differ significantly. Distribution of NKT cells was observed in the epithelium of the control of
prostate tissue significantly more than in the epithelium of prostate tissue in patients with
BPH and CaP patients. The frequency of B lymphocytes was significantly higher in the
peripheral blood of patients with BPH than the frequency of B lymphocytes in the peripheral
blood of patients with CaP and the control group. The percentage of B lymphocytes isolated
from prostate tissue of patients with BPH and patients with CAP did not differ significantly.
The percentage of perforin positive cells was statistically lower in the prostatic tissue of
patients with CaP than in patients with BPH. A smaller percentage of perforin was observed
in CD3+CD56- T lymphocytes and its CD3+CD4+CD56- and CD3+ CD8+CD56-
subpopulations, in NK cells and their CD3-CD56+ subpopulation and inNKT cells isolated
from prostate tissue of patients with CaP when compared to patients with BPH. NK cells
isolated from peripheral blood of patients with BPH and CaP patients had statistically
significant lower cytotoxic activity against K-562 sensitive cell line in comparison to NK
cells from peripheral blood of healthy volunteers. Mononuclear cells isolated from prostate
tissue of patients with BPH and CaP patients had a small cyotoxic activity against K-562
sensitive cell line. Statistically higher percentage of IFN
+ cells was found only in NK cells
isolated from patients with BPH, while the percentage of IFN
+, and IL-4+ cells did not differ
significantly in other lymphocyte subpopulations isolated from prostate tissue of patients with
BPH and CaP. A negative correlation was found between the percentages of CD3+CD56- cells in peripheral blood and prostate tissues and PSA in patients with CaP, but not in patients with
BPH. We found a positive correlation between the percentage of CD4+CD25+FOXP3+ cells in
peripheral blood and prostate tissues and PSA in patients with CaP, but not in patients with
BPH. A negative correlation was found between perforin-positive cells, CD3+perforin+,
CD56+ perforin+ cells and PSA in prostatic tissue only CaP patients.
Conclusion: The results of this study clearly indicate that a small percentage of cells with
cytotoxic phenotype, especially NK cells, a small proportion of perforin-positive cells,
including T lymphocytes, NK cells and NKT cells, the low cytotoxic activity and the increase
of T regulatory cells in patients with BPH, especially in patients with CaP is as a result of
local events in prostate tissue and one of the mechanisms involved in the pathogenesis and
progression of BPH and CaP. To confirm these results further investigations shoud be
preformed.
Keywords
Benigna prostatična hiperplazija
karcinom prostate
NK stanice
NKT stanice
perforin
regulacijske T stanice
stanična imunost
Keywords (english)
Benign Prostatic Hyperplasia
Prostate Cancer
NK Cells
NKT Cells
Perforin
Tregs
Cellular Immunity
Language croatian
URN:NBN urn:nbn:hr:188:571893
Project Number: 062-0620096-0094 Title: Regulacijske T i NKT stanice u kontroli tumorskog rasta, opeklina i autoimunosti Leader: Ines Mrakovčić-Šutić Jurisdiction: Croatia Funder: MZOS
Study programme Title: Biomedicine Postgraduate (doctoral) study programme Study programme type: university Study level: postgraduate Academic / professional title: doktor/doktorica znanosti, područje biomedicine i zdravstvo (doktor/doktorica znanosti, područje biomedicine i zdravstvo)
Catalog URL https://libraries.uniri.hr/cgi-bin/unilib.cgi?form=D1121018060
Type of resource Text
Extent 183 str.
File origin Born digital
Access conditions Closed access
Terms of use
Created on 2017-01-19 19:33:14