Title Uloga granulizina na majčino-fetalnom spoju tijekom rane trudnoće : doktorski rad Author Danijela Veljković Vujaklija Mentor Daniel Rukavina (mentor)Committee member Gordana Laškarin (predsjednik povjerenstva)Committee member Marina Ivanišević (član povjerenstva)Committee member Herman Haller (član povjerenstva)Committee member Daniel Rukavina (član povjerenstva)Granter University of Rijeka Defense date and country 2011-06-17, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 618 - Gynaecology. Obstetrics Abstract Cilj istraživanja: Granulizin je citolitičko/apoptotička molekula sposobna ubiti različite patogene i tumorske stanice. Malo je poznato o ulozi granulizina na majčino-fetalnom spoju. Cilj našeg istraživanja bio je ispitati: (1) izraženost i unutarstanični smještaj granulizina u decidui i perifernoj krvi rane trudnoće, (2) utjecaj molekula HLA-G i HLA-C na izraženost, lučenje i unutarstanični smještaj granulizina, (3) sposobnost stanica NK da izlučuju granulizin, (4) mehanizme regulacije izraženosti granulizina Th1 citokinima, antigen predočnim stanicama (APS) i progesteronom.
Materijal i metode: Uzorke decidualnog tkiva i periferne krvi dobivali smo sa Klinike za ginekologiju i porodništvo KBC-a Rijeka nakon namjernog prekida normalne trudnoće (6.-10. tjedan). Različite stanične subpopulacije pročistili smo metodom pozitivne magnetske separacije. Izraženost granulizina u decidui i perifernoj krvi analizirali smo metodom imunohistokemije i imunofluorescencije uz očitavanje protočnim citometrom ili konfokalnim mikroskopom. Izlučivanje granulizina iz pročišćenih stanica NK ispitali smo imunoenzimskim testom ELISA. Gensku izraženost granulizina i ostalih citolitičkih medijatora analizirali smo metodom lančane reakcije polimeraze u stvarnom vremenu.
Rezultati: Granulizin je obilno izražen u decidui. Više od 85% decidualnih stanica NK izražava granulizin. Decidualni limfociti T, u odnosu na limfocite T iz periferne krvi, u značajno većem postotku izražavaju granulizin. Decidualne APS također izražavaju granulizin. Izraženost granulizina na genskoj razini u decidualnim staničnim subpopulacijama (stanice NK, NKT, limfociti T, nezrele dendritične stanice, makrofagi) je značajno viša u odnosu na ostale citolitičke medijatore (perforin, FasL, TRAIL). U usporedbi s perforinom, granulizin se u stanicama nalazi pretežno u granulama koje su smještene uz staničnu membranu dok se perforin nalazi u centralnije smještenim granulama. Kultivirane u mediju, decidualne stanice NK spontano luče velike količine granulizina. Lučenje se može potaknuti HLA-C molekulama, a potisnuti HLA-G molekulama koje potiskuju i proteinsku izraženost granulizina u stanicama NK. Decidualne APS i IL-15 također sudjeluju u kontroli izraženosti granulizina u stanicama NK.
Zaključak: Obilna izraženost granulizina u imunokompetentnim stanicama, kao i sposobnost stanica NK da spontano izlučuju velike količine granulizina upućuje na ulogu granulizina kao zaštitne i imunomodulacijske molekule na majčino-fetalnom spoju. Klasične i neklasične HLA molekule I. razreda izražene na stanicama ekstraviloznog trofoblasta sudjeluju u regulaciji lučenja i izraženosti granulizina u stanicama NK te time vjerojatno doprinose očuvanju trudnoće.
Abstract (english) Objectives: Granulysin is a novel cytolytic protein lytic against a variety of tumour cells and microbes. The role(s) of granulysin during pregnancy have not been extensively explored. The aim of our study was to examine: (1) granulysin expression and localization in the first trimester pregnancy peripheral blood (PB) and decidua, (2) the influence of HLA-G and HLA-C molecules on granulysin expression, secretion and intracellular localization, (3) the ability of NK cells to secrete granulysin, and (4) the role of antigen presenting cells (APC), Th1 cytokines and progesteron in the regulation of granulysin expression.
Material and methods: Decidual tissue samples and peripheral blood were obtained from Department of Gynecology and Obstetrics, Clinical Hospital Center, Rijeka after an elective pregnancy termination of normal 6.-10. week old pregnancies. Different cell subpopulations were purified by positive magnetic separation. Granulysin expression was analyzed using cell permeabilization method, flow cytometry, confocal microscopy and immunohistochemistry. Granulysin secretion by purified NK cells was detected by ELISA method. Granulysin, perforin, FasL and TRAIL gene expression was analyzed by quantitative real-time PCR.
Results: Granulysin is abundantly expressed at the maternal-fetal interface in early pregnancy. Over 85% of decidual NK cells express granulysin. Decidual T lymphocytes, in comparison to PB T lymphocytes, express significantly higher levels of granulysin. Decidual APC also express granulysin. In comparison to perforin and other cytolytic mediators, granulysin mRNA is present at significantly higher levels in NK cells, NKT cells, macrophages and immature dendritic cells, but not in T lymphocytes. Intracellularly, granulysin is mostly stored in the granules near the cell membrane and rarely colocalizes with perforin which is stored in more centrally positioned granules. When cultured, decidual NK cells spontaneously release high quantities of granulysin. HLA-C molecules enhance granulysin secretion, whereas HLA-G molecules suppress granulysin secretion and protein expression in NK cells. Decidual APC participate in the control of granulysin expression in NK cells as well.
Conclusion: Abundant expression of granulysin by decidual immunocompetent cells and the capacity of decidual NK cells to spontaneously secrete high quantities of granulysin points to important protective and immunomodulatory role(s) that this molecule could play at the maternal-fetal interface. Classical and non-classical HLA class I molecules expressed by extravillous trophoblast cells contribute to the maintenance of pregnancy by regulation of granulysin expression and secretion in NK cells.
Keywords
Decidua
Granulizin
Limfociti T
Mehanizmi staniĉne citotoksiĉnosti
Perforin
Stanice NK
Trudnoća
Keywords (english)
Decidua
Granulysin
Mechanisms of cell cytotoxicity
NK cells
Perforin
Pregnancy
T Lymphocytes
Language croatian URN:NBN urn:nbn:hr:188:697453 Project Number: 062-0620402-0376 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Catalog URL https://libraries.uniri.hr/cgi-bin/unilib.cgi?form=D1121018002 Type of resource Text Extent 198 str. File origin Born digital Access conditions Closed access Terms of use Repository Repository of the University of Rijeka Library Created on 2017-01-19 19:12:41
