magistrsko delo
Petra Kern (Avtor), Uroš Rajčević (Mentor)

Povzetek

Celična terapija s CAR-T se je v preteklih letih izkazala za eno najobetavnejših novih terapij za zdravljenje malignih obolenj. Zaradi uporabe bolniku lastnih (avtolognih) celic, ki predstavljajo spremenljiv dejavnik, se pojavlja potreba po in vitro testiranju aktivnosti celic CAR-T pred vnosom v pacienta. V magistrski nalogi smo s pomočjo elektroporacije v celično linijo humanih limfocitov T CD4+ vnesli receptor CAR anti-CD19 in reporterski protein GFP, nato smo dvojno pozitivne celice ločili s pomočjo FACS in tako dobili homogeno celično kulturo. Celice CAR-T smo izpostavili antigenu CD19. Gojili smo jih v kokulturi s celično linijo humanih limfocitov B, ki izražajo ta antigen. Po 48 urah kokultivacije smo v supernatantu celične kokulture s pomočjo posrednega testa ELISA določali koncentracijo citokinov IL-2, TNF-alfa in IFN-gama. Citokini v supernatanu kokulture so pokazatelj aktivacije celic CAR-T z antigenom CD-19. Celice smo prešteli s pomočjo Bürker – Türkove števne komore pod fluorescentnim in pod svetlobnim mikroskopom, da bi ugotovili, kako se je spremenilo število celic in razmerje med efektorskimi (celice CAR-T, ki zeleno fluorescirajo) in tarčnimi celicami (limfociti B, ki ne fluorescirajo). Z optimalnimi pogoji elektroporacije smo dosegli, da je 22,1 % živih celic izražalo CAR in GFP. V 48 urah kokultivacije se razmerje med efektorskimi in tarčnimi celicami ni spremenilo, prav tako se ni spremenila končna skupna koncentracija celic. Statistično značilno razliko v izločanju citokinov IL-2 in TNF-alfa smo opazili pri razmerju efektorskih in tarčnih celic 1:1. Izločanja IFN-gama z našim testom nismo zaznali. Pokazali smo, da je kokultivacija celic CAR-T skupaj s specifičnim antigenom povzročila izločanje citokinov, ki jih lahko kvantitativno določimo z indirektnim testom ELISA in s tem posredno ovrednotimo pričakovano učinkovitost terapije.

Ključne besede

celična terapija;CAR-T;limfociti;elektroporacija;ELISA;citokini;genotoksičnost;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UL BF - Biotehniška fakulteta
Založnik: [P. Kern]
UDK: 606:616-006.6:602.68:602.621(043.2)
COBISS: 72139779 Povezava se bo odprla v novem oknu
Št. ogledov: 529
Št. prenosov: 120
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Introduction of functional CAR-T activity tests in vitro
Sekundarni povzetek: CAR-T cell therapy is one of the most promising new therapies for treatment of malignancies. Because of the use of autologous cells, which are a variable factor, there is a need for in vitro testing of CAR-T cell activity prior to aplication to patient. In the thesis, electroporation was used to insert anti-CD19 CAR receptor and reporter protein GFP into CD4+ human lymphocyte cell line. Next, double positive cells were sorted using fluorescence-assisted cell sorting to acquire a homogenous cell culture. CAR-T cells were then exposed to the antigen CD19 present on human B-cell line. We cocultured both cell lines for 48 hours. Using an indirect ELISA test, we then determined the concentration of cytokines IL-2, TNF-alpha, and IFN-gamma in the cell culture supernatant. Cytokines in the coculture supernatant indicate that activation of CAR-T cells via CD-19 antigen was successful. Cells were counted using Bürker – Türk counting chamber to determine how the number of cells in the culture and the ratio between effector (fluorescent CAR-T cells) and target cells (B-cells) has changed. With optimised electroporation parameters we accomplished expression of CAR and GFP proteins in 22,1 % of live cells. In the 48 hours of cocultivation the ratio between effector and target cells did not change. The density of cell culture did not differ between test and control. Statistically significant difference in cytokine IL-2 and TNF-alpha secretion was observed in effector and target cell ratio of 1:1. Secretion of IFN-gamma cytokine was not detected. We showed that cocultivating CAR-T cells with their specific antigen causes activation of the cells and secretion of cytokines which can be quantitatively measured using indirect ELISA test. The results can be used to indirectly predict the efficacy of cell therapy.
Sekundarne ključne besede: cell therapy;lymphocytes;electroporation;cytokines;
Vrsta dela (COBISS): Magistrsko delo/naloga
Študijski program: 0
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Strani: XI, 50 f.
ID: 13150391