Title Morfogenetska i imunomodulacijska svojstva stresnih proteina metalotioneina i glikoproteina 96 tijekom regeneracije jetre u miša : doktorska disertacija Author Hrvoje Jakovac Mentor Biserka Radošević-Stašić (mentor)Committee member Čedomila Milin (predsjednik povjerenstva)Committee member Tanja Marotti (član povjerenstva) Committee member Biserka Mulac-Jeričević (član povjerenstva)Committee member Sandra Milić (član povjerenstva)Committee member Biserka Radošević-Stašić (član povjerenstva)Granter University of Rijeka Defense date and country 2009-02-26, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 616 - Pathology. Clinical medicine Abstract Cilj istraživanja:
Cilj istraživanja bio je utvrditi obrazac izražaja metalotioneina (MT I+II) i proteina
toplinskog šoka Gp96 na genskoj i proteinskoj razini u procesima proliferacije i
diferencijacije stanica tijekom normalnog rasta i embriogeneze, njihov izražaj u limfatickim
organima, te pratece promjene fenotipskih i funkcionalnih karakteristika limfatickih stanica, u
razlicitim vremenskim intervalima tijekom regeneracije jetre nakon jednotrecinske parcijalne
hepatektomije. Buduci da je izražaj metalotioneina usko vezan uz razinu tkivnih metala,
nastojale su se ispitati i promjene njihovih koncentracija tijekom regeneracije jetre.
Osim toga, analiziran je izražaj metalotioneina i Gp96 na fetomaternalnom spojištu i
placentarnim tkivima tijekom normalnog rasta i razvoja u sklopu singenicne trudnoce, kako bi
se utvrdio njihov znacaj u procesima placentacije.
Materijali i metode:
Istraživanja su provedena na C57/Bl6 soju miševa starosti 2 - 3 mjeseca. Promjene
proteinskog izražaja utvrdene su imunohistokemijski na parafinskim tkivnim rezovima,
odnosno citospin pripravcima, uporabom monoklonskih protutijela i DAKO EnVision +
System kita. Indukcija MT-I gena utvrdena je metodom reverzne transkripcije i klasicne
lancane reakcije polimerazom, dok su promjene transkripcijske aktivnosti Gp96 gena
ustanovljene kvantitativnom lancanom reakcijom polimerazom u realnom vremenu.
Apoptoticne stanice detektirane su TUNEL metodom na parafinskim tkivnim rezovima.
Koncentracije tkivnih metala odredene su spektrometrijski. Imunofenotipizacija limfatickih
stanica izvedena je metodom izravne imunofluorescencije i analizom protocnim citometrom,
kojim su detektirane i mrtve stanice u sklopu funkcijskog testa citotoksicnosti.
Fetalna i placentarna tkiva analizirana su 16. dana gestacije. Rezultati:
Nakon jednotrecinske parcijalne hepatektomije utvrden je brzi porast izražaja stresnih
proteina metalotioneina i glikoproteina 96 u regenerirajucoj jetri, slezeni, timusu i koštanoj
srži, uz promjene transkripcijske aktivnosti MT-I i Gp96 gena. Istovremeno je zabilježen i
porast koncentracija Zn2+, Ca2+, Mg2+ i Fe2+ iona u tkivu regenerirajuce jetre, pracen padom
koncentracija ovih metala u slezeni, te padom koncentracije Zn2+ u timusu. Podrucja
regenerirajuce jetre u kojih je ustanovljeno prisustvo apoptoticnih hepatocita pokazivala su
inverzan odnos sa podrucjima u kojima je bio prisutan snažan izražaj MT I+II proteina.
Porast izražaja stresnih proteina MT I+II i Gp96 nakon parcijalne hepatektomije bio je
pracen porastom udjela aktiviranih NKT stanica u jetri, T regulacijskih stanica u jetri i timusu,
te aktiviranih dendritickih stanica u jetri i slezeni. Nadalje, utvrdeno je da je parcijalnom
hepatektomijom potaknut izražaj TLR2 u slezeni i timusu, gdje je ujedno znacajno porastao
udio timocita koji podliježu apoptozi. Istodobno, mononuklearne limfaticke stanice jetre i
slezene stekle su veci stupanj NKT i NK citotoksicne aktivnosti.
Izražaj MT I+II i Gp96 proteina bio je prisutan u mnogim fetalnim tkivima, napose u
epitelnima, kao i u podrucju fetomaternalnog spojišta.
Zakljucak:
Porast izražaja MT I+II i Gp96 proteina u tkivu jetre i limfatickih organa nakon
parcijalne hepatektomije, kao i njihovo prisustvo u fetalnim i placentarnim tkivima, ukazuju
na ulogu ovih proteina u proliferacijskim zbivanjima tijekom tkivnog rasta, te u indukciji i
aktivaciji autoreaktivnih klonova stanica, kao što su NKT i T regulacijski limfociti, koji bi
mogli sudjelovati u kontroli reparacijskog procesa i kompenzatornog rasta, ali i u održanju
imunološke tolerancije nakon tkivnog oštecenja.
Abstract (english) Objectives:
The aim of this study was to assess the expression pattern of methalothioneins
(MT I+II) and heat shock protein Gp96 at gene and protein level during proliferative and
differentiation processes in the course of normal growth and embryogenesis, their expression
in lymphatic organs as well as the concomitant phenotypical and functional changes of
lymphatic cells, at different time points during the liver regeneration induced by one-third
partial hepatectomy. Since metallothionein expression is closely related to metal tissue
content, during the regenerative process we examined the tissue concentrations of Zn2+, Ca2+,
Mg2+ and Fe2+.
Additionally, metallothionein and Gp96 expression were analyzed on feto-maternal
interface and placental tissues during normal growth and development in context of syngeneic
pregnancy, in order to appoint their importance in placentation processes.
Materials and methods:
The study was performed on mice of strain C57/Bl6, aged 2-3 months. Protein
expression was determined by immunohistochemistry on paraffin-embedded tissue sections
and citospin preparations using monoclonal antibodies and DAKO EnVision + System kit. In
order to examine MT-I gene induction, reverse transcription polymerase chain reaction was
done, whereas transcriptional changes of Gp96 gene were assessed by quantitative real-time
polymerase chain reaction. Apoptotic cells were detected by TUNEL method. Tissue metal
concentrations were investigated spectrometricaly. Phenotypic analyses of mononuclear
lymphatic cells were performed by direct immunofluorescent staining and flow cytometry,
which was also used to detect death cells during functional cytotoxicity assay. Results:
The data revealed that one-third partial hepatectomy leads to high overexpression of
stress proteins metallothioneins and Gp96 in regenerating hepatocytes, as well as in spleen,
thymus and bone marrow, accompanied by transcriptional changes of MT-I and Gp96 genes.
Simultaneously, accumulation of Zn2+, Ca2+, Mg2+ and Fe2+ ions was noticed in the
regenerating liver, while there was a concurrent decrease of all these metals in the spleen, and
the concentration of Zn2+ in the thymus. The areas containig apoptotic hepatocytes in the
regenerating liver inversely correlated with those overexpressing MT I+II proteins.
MT I+II and Gp96 upregulation after partial hepatectomy was followed by
accumulation of activated NKT cells in the liver and increased proportion of regulatory T
cells in the liver and thymus, as well as activated dendritic cells in the liver and spleen. In
addition, TLR2 induction after partial hepatectomy was observed in the spleen and thymus,
where the increased proportion of apoptotic thymocytes was also found. Simultaneously,
intrahepatic and splenic mononuclear lymphatic cells acquired enhanced NKT and NK
cytotoxic activity.
The data also showed that MT I+II and Gp96 expression is highly upregulated during
embryogenesis, particularly on several epithelial tissues, as well as at area of feto-maternal
interface.
Conclusion:
Upregulation of MT I+II and Gp96 proteins in liver and lymphatic organs following
partial hepatectomy, and their presence in fetal and placental tissues, points out the role of
these proteins in proliferative processes during tissue growth, as well as in the recruitment and
activation of autoreactive clones of cells, such are NKT and regulatory T lymphocytes, that
might participate in the control of reparatory processes and compensatory growth, and in the
maintainance immunological tolerance after tissue injury.
Keywords
Gp96
Hepatektomija
parcijalna
Metalotioneini
NK stanice
NKT stanice
Regeneracija jetre
Regulacijske T stanice
Keywords (english)
Gp96
Hepatectomy
partial
Liver regeneration
Metallothioneins
NK cells
NKT cells
Regulatory T cells
Language croatian URN:NBN urn:nbn:hr:188:641335 Project Number: 062-0621341-1337 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Catalog URL http://libraries.uniri.hr/cgi-bin/ucat/unilib.cgi?form=D1120228016 Type of resource Text Extent 201 str. File origin Born digital Access conditions Open access Terms of use Created on 2017-01-19 18:45:11
