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Celična imunoterapija je trenutno eno najobetavnejših, hitro razvijajočih terapevtskih področij. Himerni antigenski receptor (CAR) je sintezni molekulski konstrukt, ki je z različnimi metodami genskega transferja vstavljen v limfocite T. To jim omogoča vezavo antigenov, tarčnih receptorjev in uničenje celic zaradi česar so uporabljeni za zdravljenje B-celičnih limfomov in levkemij, hitro pa se razvijajo tudi himerni antigenski receptorji proti drugim ciljem. Glavni namen magistrskega dela je bila uporaba elektrogenskega transferja za izražanje molekule CAR specifične za označevalec CD19 v limfocitih T ter njihovo namnoževanje in karakterizacija. Namnožili smo plazmid z zapisom za GFP, s katerim smo optimizirali pogoje eletkroporacije, ter plazmida z zapisom za CAR in encim transpozazo. Karakterizacijo celic smo opravili z opazovanjem pod fluorescenčnim mikroskopom in pretočno citometrijo. Kot optimalni pogoj za transfekcijo limfocitov T se je izkazal en pulz dolžine 15 ms pri napetosti 2400 V. Izražanje molekulskega konstrukta CAR smo dokazali s pretočno citometrijo in funkcijskim testom ELISA, s katerim smo določili koncentracijo izločenega IL-2 ob celični aktivaciji po vezavi receptorja CAR in tarčnega označevalca CD19. Cell immunotherapy is one of the most promising, fastest growing therapeutic treatments. Chimeric antigen receptor (CAR) is a synthetic molecular construct, which is inserted in T cells by using different methods of gene transfer. That allows lymphocytes T to bind antigens and other receptors and destroy select cells, which makes them excellent for treatment of B-cell leukemias and lymphomas. There has been a rapid development of chimeric antigen receptors that target other diseases. Main goal of the master's thesis is use of electrogenic transfer for expression of CD19 specific CAR molecules in lymphocytes T and their proliferation and characterization. We propagated a plasmid carrying a GFP gene, which we used for optimizing the conditions of electroporation, as well as a set of plasmids carrying a CAR gene and gene for the enzyme transposase. We characterized the cells by using a fluorescent microscope and flow cytometry. Optimal transfection condition for lymphocytes T was as follows – one pulse with a duration of 15 ms at 2400 V. Expression of CAR was proven with flow cytometry and a functional ELISA test, that was used for measuring the concentration of secreted interleukin 2 following cell activation. |
