Title Izražaj stresnih bjelančevina metalotioneina i glikoproteina 96 u štakorskih sojeva različite sklonosti za razvoj eksperimentalnog autoimunosnog encefalomijelitisa Title (english) EXPRESSION OF STRESS PROTEINS METALLOTHIONEINS AND GLYCOPROTEIN 96 IN RAT STRAINS WITH DIFFERENT SUSCEPTIBILITY TO EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS Author Tanja Grubić Kezele Mentor Hrvoje Jakovac (mentor)Committee member Smiljana Ristić (član povjerenstva)Committee member Alenka Gagro (član povjerenstva)Committee member Lidija Tuškan-Mohar (član povjerenstva)Committee member Hrvoje Jakovac (član povjerenstva)Granter University of Rijeka Defense date and country 2015, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 616 - Pathology. Clinical medicine ThesaurusMESH - Medical Subject Headings )
Encephalomyelitis, Autoimmune, Experimental
Glycoproteins
Metallothionein
Multiple Sclerosis
Abstract Cilj istraživanja:
Cilj istraživanja je bio utvrditi postoje li razlike u konstitutivnom i inducibilnom izražaju stresnih bjelančevina metalotioneina I+II (MT I+II) i bjelančevine toplinskog šoka glikoproteina 96 (Gp96) između štakora DA i AO soja tijekom razvoja eksperimentalnog autoimunosnog encefalomijelitisa. Pošto su citokini IL-6 i TGF-β (od engl. transforming growth factor β), kao i transkripcijski čimbenik NF-kB (od. engl. nuclear factor kappa-light-chain-enhancer of activated B cells), važni za odvijanje patogeneze multiple skleroze i EAE-a, istovremeno su se ispitivale pridružene razlike njihovog izražaja u spomenutim tkivima. Budući da je izražaj MT I+II usko vezan uz tkivne metale, ispitivale su se razlike u tkivnim sadržajima iona cinka i bakra.
Materijali i metode:
Istraživanja provedena su na mužjacima štakora AO i DA soja, starosti 2-3 mjeseca. Uzorci za analizu uključivali su tkivo jetre, velikog i malog mozga i zasebno hipokampusa iz intaktnih neimuniziranih životinja te imuniziranih životinja žrtvovanih u predkliničkoj fazi bolesti (7. dan) i trenutku najintenzivnije kliničke slike (12. dan). Promjene izražaja bjelančevina utvrđene su imunohistokemijskim bojanjem na parafinskim tkivnim rezovima te Western blot analizom, uporabom odgovarajućih protutijela. Indukcija MT-I gena utvrđena je metodom reverzne transkripcije u realnom vremenu. Koncentracije tkivnih metala određene su spektrofotometrijski, a tkivni sadržaj i distribucija slobodnih cinkovih iona prikazani bojanjem na parafinskim tkivnim rezovima uporabom specifičnog fluorescentnog kelatora cinkovih iona Zinquina. Dodatna obrada rezultata je uključivala kvantifikaciju inteziteta imunohistokemijskoh bojanja, inteziteta bojanja Zinquinom te intenziteta signala bendova dobivenih Western blotom u odgovarajućim softverskim programima.
Rezultati:
Rezultati su pokazali veći konstitutivni izražaj MT I+II u hipokampusu, malom mozgu i jetri štakora AO soja u odnosu na DA soj. Izražaj MT I+II nije se znatno mijenjao nakon imunizacijskog protokola za indukciju EAE-a. U štakora DA soja je utvrđena znatna indukcija MT I+II u istim tkivima nakon imunizacije. Transkripcijska aktivnost MT-I gena ponašala se prema identičnom obrascu u oba soja. Prateći porast izražaja MT I+II nakon indukcije EAE-a, u DA soja povećala se i ukupna koncentracija tkivnog cinka u navedenim tkivima, posebice u jetri, gdje je utvrđen i porast bakra, dok se količina slobodnih cinkovih iona u mozgu i jetri štakora DA soja nije znatno mijenjala nakon imunizacijskog protokola. U AO soja sadržaj slobodnih cinkovih iona porastao je nakon imunizacije u hipokampusu i jetri, dok se ta razina značajno smanjila u malome mozgu. Izražaji citokina TGF-β i IL-6 ponašali su se prema obrascu izražaja MT I+II u oba soja. Utvrđen je viši konstitutivni izražaj transkripcijskog čimbenika NF-kB u jetri štakora AO soja, u odnosu na DA soj, koji je značajno snižen nakon imunizacije u štakora AO soja. U mozgu intaktnih štakora oba soja nije utvrđena znatna razlika tog čimbenika, te je on ostao gotovo nepromijenjen nakon imunizacije u štakora AO soja, a u hipokampusu DA soja se znatno povisio. Izražaj bjelančevine toplinskog šoka Gp96 pokazao je sličnost s izražajima MT I+II i citokina u štakora AO soja, dok se u DA soja izražaj istoga smanjio u dentatnom girusu hipokampusa nakon imunizacije.
Zaključci:
Između štakora DA i AO soja postoje razlike u inducibilnosti i konstitutivnom izražaju stresnih bjelančevina MT I+II i Gp96, što doprinosi različitoj sklonosti razvoju EAE-a.
Osim toga, različitoj sklonosti, barem djelomično, doprinose i različiti izražaji citokina TGF-β i IL-6, kao i transkripcijskog čimbenika NF-kB u mozgu i jetri, molekula s dobro poznatom ulogom u upalnim i imunosnim procesima.
Abstract (english) Objectives:
The aim of this study was to determine differences of constitutive and inducibile expression of stress proteins metallothioneins I+II (MT I+II) and heat shock protein glycoprotein 96 (Gp 96) between DA and AO rat strains which were subjected to immune protocol for induction of experimental autoimmune encephalomyelitis (EAE). Concomitantly, the difference of expression of cytokines IL-6 and TGF-β as well as transcription factor NF-kB was examined, since it is known that they have important role in pathogenesis of multiple sclerosis and EAE. Considering that MT I+II are closely related to tissue metals, concentrations of Zn and Cu ions were additionally assessed in the studied tissues.
Materials and methods:
The study was performed on male AO and DA rat strains, aged 2-3 months. The samples included hepatic, cerebral, cerebellar and hippocampal tissue from intact (nonimmunized) and immunized animals sacrificed in preclinical (7th day) phase of disease as well as in the moment of the most intensive clinical symptoms (12th day). Protein expression was determined by immunohistochemistry on paraffin-embedded tissue sections and by Western blot analysis using antibodies of interest. The gene expression of MT-Ia was analyzed by absolute quantitative real-time polymerase chain reaction. Tissue metal concentrations were determined spectrometricaly, while tissue content and distribution of free zinc ions was determined using ion chelator Zinquin. The intensity of immunohistochemical and Zinquin staining and Western blot bends was quantficated using computer software.
The results showed higher constitutive hippocampal, cerebellar and hepatic expression of MT I+II proteins in AO rats in comparison to DA rats. Expression of MT I+II did not change significantly after immunization protocol for EAE induction in AO rats. In DA rats, high induction of MT I+II proteins was found after immunization in all analyzed tissues. Transcriptional activity of MT-Ia gene correlated with protein expression in both rat strains. Concurrently with MT I+II expression in DA rats after EAE induction, totally tissue zinc concentration has also increased, especially in liver, where increase of total copper concentration has also been observed. Furthermore, brain and hepatic level of free zinc ion did not change significantly after immunization. Hippocampal and hepatic level of free zinc ions increased, but cerebellar level decreased after immunization in AO rats. TGF-β and IL-6 expression correlated with MT I+II expression in both rat strains. Furthermore, higher constitutive hepatic NF-kB expression was found in AO rats in comparison to DA rats, and expression decreased significantly after immunization. No differences of NF-kB expression was found between brain tissues of both intact rat strains, but after immunization this transcription factor was significantly upregulated in hippocampus of DA rats, while in AO strain remained at a basal level. Expression of heat shock protein Gp96 showed similarity with MT I+II and cytokines expressions in AO rats, but this expression decreased in hippocampal dentate gyrus in DA rats after immunization.
Conclusions:
There is a different constitutive and inducible expression of stress proteins MT I+II and Gp96 between AO and DA rats, suggesting their role in different susceptibility to immune mediated encephalomyelitis. Moreover, different sensitivity could also be, at least partly, attributed to different expression of TGF, IL-6 and NF-kB, molecules with well-known role in the inflammatory and immune processes.
Keywords
Eksperimentalni autoimunosni encefalomijelitis
Glikoprotein 96
Metalotioneini
Multipla skleroza.
Keywords (english)
Experimental autoimmune encephalomyelitis
Glycoprotein 96
Metallothioneins
Multiple sclerosis.
Language croatian URN:NBN urn:nbn:hr:184:888813 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Created on 2016-02-17 16:13:40
