Title Uloga molekule CD26 u razvoju kolitisa u miša : disertacija Title (english) Role of CD26 molecule in the colitis development in the mice Author Dijana Detel Mentor Jadranka Varljen (mentor)Committee member Zlatko Trobonjača (predsjednik povjerenstva)Committee member Željko Kućan (član povjerenstva)Committee member Brankica Mijandrušić-Sinčić (član povjerenstva)Granter University of Rijeka Defense date and country 2009-08-26, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 616 - Pathology. Clinical medicine Abstract Upalne bolesti crijeva su kompleksne, kronične, imunološki posredovane bolesti obiljeţene ponavljajućim upalnim procesima koji rezultiraju oštećenjem i gubitkom funkcije crijevne sluznice. Dosadašnja saznanja ukazuju na povezanost aktivacije imunološkog odgovora s promjenom aktivnosti i izraţaja molekule CD26/DPP IV.
Cilj istraţivanja bio je ispitati je li i na koji način molekula CD26/DPP IV uključena u lokalne i sistemske mehanizme koji dovode do oštećenja sluznice i razvoja upalne bolesti crijeva u in vivo modelu kolitisa uspostavljenog u CD26 deficijentnom (CD26-/-) i divljem tipu miša (C57BL/6). Kako bi se ispitao utječe li nedostatak molekule CD26/DPP IV na razvoj kolitisa, pratio se razvoj, intenzitet i cijeljenje kolitisa u različitim vremenskim intervalima temeljem kliničkih i histoloških parametara na lokalnoj i sistemskoj razini u obje skupine miševa. Budući da je oštećenje sluznice posredovano imunološkom reakcijom, pratio se izraţaj specifičnih antigena na lokalnoj razini te fenotipske promjene limfatičnih stanica slezene, jetre i periferne krvi tijekom razvoja i cijeljenja kolitisa. Određene su dinamičke promjene aktivnosti CD26/DPP IV tijekom razvoja kolitisa u serumu te sluznici tankog i debelog crijeva C57BL/6 miševa.
Materijal i metode: Istraţivanje je provedeno na dvjema vrstama laboratorijskih miševa: divlji tip miša soja C57BL/6, te miševi kojima je inaktiviran gen za molekulu CD26 (C57BL/6 Jbom-ob, CD26-/-), istog spola i dobi, starosti od 10 do 12 tjedana. Model induciranog kolitisa izazvan je u obje ispitivane skupine miševa primjenom 3% (w/v) otopine natrijevog dekstran-sulfata (DSS) per os kroz sedam dana. Razvoj kolitisa i procjena intenziteta bolesti u svakoj ispitivanoj skupini ţivotinja pratio se temeljem kliničkih simptoma i izračuna indeksa aktivnosti bolesti. Stupanj oštećenja sluznice tankog i debelog crijeva pratio se određivanjem mikroskopskog indeksa oštećenja te histomorfometrijskom analizom u različitim vremenskim intervalima tijekom razvoja i cijeljenja kolitisa na parafinskim tkivnim rezovima. Imunofenotipizacija limfatičkih stanica izvedena je metodom izravne imunofluorescencije i analizom protočnim citometrom. Promjene izraţaja specifičnih staničnih biljega utvrđene su imunohistokemijski na parafinskim i/ili smrznutim tkivnim rezovima. Aktivnost serumske i intestinalne CD26/DPP IV u C57BL/6 miševa određena je spektrofotometrijski. Rezultati: Razvoj kolitisa u obje ispitivane skupine miševa rezultirao je statistički značajnim padom tjelesne mase, promjenom duţine i mase debelog crijeva te porastom mase slezene i indeksa aktivnosti bolesti. U skupini C57BL/6 miševa maksimalni pad tjelesne mase zabiljeţen je sedmog dana i praćen je statistički značajnim skraćenjem debelog crijeva i porastom mase slezene. Oporavak započinje devetog, a završava petnaestog dana. U skupini CD26-/- miševa, maksimalni pad tjelesne mase zabiljeţen je devetog dana, a statistički značajno skraćenje debelog crijeva sedmog dana. Oporavak započinje desetog, a završava dvadesetog dana. Razvoj kolitisa u obje ispitivane skupine miševa praćen je histomorfometrijskim promjenama sluznice debelog crijeva odnosno statistički značajnim smanjenjem broja i dubine te povećanjem širine kripti.
U usporedbi s C57BL/6 miševima, CD26-/- miševi imaju sniţen udio CD4+ stanica u slezeni i NK stanica u jetri te stastistički značajno veći udio CD8+ stanica u jetri. Tijekom razvoja kolitisa dolazi do akumulacije NK stanica u slezeni i jetri CD26-/- miševa te NKT stanica u slezeni i jetri obje ispitivane skupine miševa. U akutnoj fazi kolitisa zabiljeţen je porast udjela CD8+ stanica u slezeni i jetri CD26-/- miševa te CD4+ stanica u jetri obje ispitivane skupine miševa. Fenotipske promjene su popraćene porastom izraţaja CD4+, CD8+ stanica i makrofaga u akutnoj fazi kolitisa u sluznici tankog i debelog crijeva obje ispitivane skupine miševa.
Zaključak: Temeljem utvrđenih statistički značajnih promjena kliničkih i histoloških parametara na lokalnoj i sistemskoj razini, model DSS induciranog kolitisa uspostavljen je u obje ispitivane skupine miševa. Tijekom razvoja i cijeljenja kolitisa nedostatak molekule CD26 utjecao je na vremenski tijek, ali ne i na intenzitet kolitisa. Utvrđene sistemske i lokalne imunološke promjene ukazuju da izraţaj i proteolitička aktivnost molekule CD26/DPP IV nemaju presudnu i jedinstvenu ulogu u procesu aktivacije i modulacije imunološkog odgovora tijekom razvoja i cijeljenja kolitisa u miša.
Abstract (english) Inflammatory bowel diseases are complex, chronic, immune-mediated diseases characterized by relapsing inflammatory processes which result in mucosa damage and loss of function. Previous studies indicate a relationship between the immune response and changes in expression and activity of CD26/DPP IV.
The aim of this study was to investigate is it and in what way the CD26 molecule involved in local and system mechanisms leading to mucosa damage and development of inflammatory bowel disease in a in vivo colitis model established in CD26 deficient (CD26-/-) and wild type mice (C57BL/6). In order to determine does it the deficiency of molecule CD26 influence colitis development, intensity and healing in different time schedule based on clinical and histological parameters on local and system level in both mice group were determined. Since mucosal damage is mediated by immune response, the expression of specific antigens at local level and phenotypic changes of lymphatic cells from spleen, liver and peripheral blood during colitis development and healing have been determined. Dynamic changes in CD26/DPP IV activity in serum and intestinal mucosa during colitis development in C57BL/6 mice have been investigated.
Materials and Methods: The research was performed using two mice strains: wild type mice strain C57BL/6 and mice with inactivated gene for molecule CD26 (C57BL/6 Jbom-ob, CD26-/-), same gender and age, aged from 10 to 12 weeks. The colitis induced model was established in both mice strains using 3% (w/v) sodium dextran sulphate solution (DSS) per os during 7 days. Colitis development and determination of severity was monitored in each group of experimental animals by clinical symptoms and calculation of disease activity index. The grade of intestinal mucosa damage was assessed by determination of microscopic damage index and by histomorphometrical analysis on paraffin sections in different time periods during colitis development and healing. The immunophenotypization of lymphatic cells was performed by direct immunofluorescence and by FACS analysis. Changes in expression of specific cell markers were determined by immunohistochemical staining methods on paraffin and/or frozen tissue sections. Serum and intestinal CD26/DPP IV activities in C57BL/6 mice were determined spectrophotometrically. Results: Colitis development in both mice strains resulted in statistically significantly in body weight decrease, changes in colon length and weight, increase of spleen mass and disease activity index. The most intensive body weight decrease was noticed the seventh day in C57BL/6 mice and was followed by a statistically significant colon shortening and increased spleen mass. The recovery starts the ninth day and ends the fifteenth day. In CD26-/- mice the most intensive body weight decrease was noticed the ninth day and the statistically significant colon shortening the seventh day. The recovery starts the tenth day and ends the twentieth day. Colitis development in both mice groups is followed by histomorphometrical changes of colon mucosa and statistically significant decrease of number and depth and increase of crypts width.
The percentage of CD4+ cells in the population of mononuclear splenic cells and NK cells in liver was decreased in CD26-/- mice in comparison with C57BL/6 mice. Furthermore, a statistically significant increase of CD8+ cells was noticed in liver of CD26-/- mice. In the acute phase of colitis, an increase of CD8+ cells ratio was noticed in liver and spleen of CD26-/- mice and CD4+ cells in liver in both mice strains. Phenotypical changes were followed by an increase in ratios of CD4+, CD8+ cells and macrophages in ileum and colon of both mice strains. An accumulation of NK cells was noticed in spleen and liver of CD26-/- mice while NKT cells were noticed to accumulate in spleen and liver of both mice strains.
Conclusion: Based on determined statistically significant changes of clinical and histological parameters at local and system level, a DSS-colitis model was established in both analyzed mice strains. The CD26 molecule deficiency influenced on time course but not on colitis severity at local level during colitis development and healing. Determined systemic and local immunological changes indicate that the expression and proteolytic activity of CD26/DPP IV do not have a crucial role in colitis development and healing in mice.
Keywords
Dipeptidil-peptidaza IV (CD26/DPP IV)
Molekula CD26
Pokusni model
Ulcerozni kolitis
Keywords (english)
Dipeptidyl peptidase IV (CD26/DPP IV)
Experimental model
Molecule CD26
Ulcerative colitis
Language croatian URN:NBN urn:nbn:hr:188:409696 Project Number: 062-0061245-0213 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Catalog URL https://libraries.uniri.hr/cgi-bin/unilib.cgi?form=D1120529073 Type of resource Text Extent 130 str. File origin Born digital Access conditions Closed access Terms of use Created on 2017-01-19 18:10:33
