magistrsko delo
Kaja Zajc (Author), Rok Kostanjšek (Reviewer), Urban Švajger (Mentor)

Abstract

Metoda ELISPOT (angl. Enzyme-Linked ImmunoSpot) se zaradi preproste izvedbe in visoke občutljivosti pogosto uporablja v diagnostiki in raziskavah na različnih področjih. Test interferon (IFN)-γ ELISPOT omogoča štetje antigensko (Ag)-specifičnih limfocitov T po Ag-specifični stimulaciji. Glavni namen magistrskega dela je bil vzpostaviti test na osnovi metode ELISPOT za določanje Ag-specifičnega imunskega odziva CD8+ citotoksičnih limfocitov T (CTL). Iz levkocitnih pripravkov zdravih krvodajalcev smo izolirali enojedrne celice periferne krvi (PBMC), ki smo jih uporabili za različne poskuse. Izvedli smo metodo mešane levkocitne reakcije (MLR), PBMC ex vivo stimulirali z različnimi peptidnimi mešanicami ali iz PBMC z imunomagnetno selekcijo izolirali celokupne CD8+ limfocite T in monocite. Slednje smo diferencirali v dendritične celice (DC), ki smo jih nadalje uporabili za in vitro stimulacijo avtolognih CD8+ CTL. Na podlagi rezultatov optimizacije metode ELISPOT smo določali Ag-specifičnost limfocitov T. Pokazali smo, da lahko z metodo ELISPOT zaznamo alo-Ag-specifične limfocite T in virusno-specifične limfocite T v populaciji PBMC po 24-urni ex vivo stimulaciji z mešanico peptidov citomegalovirusa (CMV), virusa Eppstein-Barr in virusa gripe A (CEF) ter mešanico peptidov CMV fosfoproteina 65 (pp65). Dokazali smo tudi, da lahko z metodo ELISPOT zaznamo redke klone melanomsko-specifičnih CD8+ CTL po in vitro stimulaciji z zrelimi avtolognimi DC (αDC in zDC), pulziranimi s peptidi, značilnimi za melanom, gp100, Melan-A in tyr. V sklopu in vitro stimulacije CD8+ CTL z DC smo dokazali tudi, da imajo zrele zDC ojačano sposobnost prožitve melanomsko-specifičnega imunskega odziva CTL v primerjavi z αDC.

Keywords

ELISPOT;IFN-γ;biologija;dendritične celice;limfociti T;melanom;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [K. Zajc]
UDC: 576
COBISS: 115668227 Link will open in a new window
Views: 129
Downloads: 31
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Other data

Secondary language: English
Secondary title: Determination of antigen-specific immune response of CD8+ cytotoxic T cells using ELISPOT technique
Secondary abstract: The ELISPOT (angl. Enzyme-Linked ImmunoSpot) method is a highly sensitive and user-friendly method, commonly applied in different areas of research and diagnostics. The interferon (IFN)-γ ELISPOT assay is used to enumerate antigen (Ag)-specific T cells upon Ag-specific stimulation. The principal aim of our study was to establish an ELISPOT assay for the determination of Ag-specific immune response of CD8+ cytotoxic T cells (CTL). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples obtained from healthy human blood donors. PBMCs were used to run the mixed lymphocyte reaction (MLR) and were ex vivo stimulated with different peptide pools. Whole CD8+ T cells and monocytes were isolated from PBMC by positive immunomagnetic selection and monocytes were used for dendritic cell (DC) differentiation. The latter were used for in vitro stimulation of autologous CD8+ CTL. After its optimization, the ELISPOT assay was applied to determine the Ag-specific immune response of T cells. Our results show that the ELISPOT assay enables the detection of allo-Ag-specific T cells and can be used to determine virus-specific T cells in PBMCs upon 24 h ex vivo stimulation with peptide pool from cytomegalovirus (CMV), Epstein-Barr virus and influenza A virus (CEF) and peptide pool of the phospohoprotein 65 (pp65) from CMV. The ELISPOT method also enables the detection of rare clones of melanoma-specific CTL upon in vitro stimulation with autologous mature DC (αDC and zDC), pulsated with melanoma-associated peptides gp100, Melan-A and tyr. Finally, our results demonstrate zDC are superior in their capacity to induce melanoma-specific CTL compared to αDC.
Secondary keywords: biology;dendritic cells;T cells;melanoma;master thesis;Citologija;Melanom;Univerzitetna in visokošolska dela;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Oddelek za biologijo
Pages: XI, 61 f., [1] f. pril.
ID: 15907817