Izražanje mRNA himernih antigenskih receptorjev v limfocitih T

• Main Author: Plesnik, Klavdija
• Other Authors: Rajčević, Uroš
• Format: Master Thesis
• Language: Slovenian
• Published: 2021
• Subjects: Limfociti T; himerni antigenski receptor; elektroporacija; mRNA; Lymphocytes T; chimeric antigen receptor; electroporation; sami
• Online Access: https://repozitorij.uni-lj.si/IzpisGradiva.php?id=125470; https://repozitorij.uni-lj.si/Dokument.php?id=141192&dn=; https://plus.si.cobiss.net/opac7/bib/55931395?lang=sl

Biomedicinska uporabnost gensko spremenjenih imunskih celic (predvsem limfocitov T) izkazuje veliko vrednost za zdravljenje malignih bolezni. V limfocitih T se izražajo različni molekulski konstrukti za terapevtske učinkovine, ki lahko vplivajo na potek bolezni. Za vstavljanje mRNA molekulskih konstruktov himernih antigenskih receptorjev (CAR T), ki smo jih v celice celičnih linij Jurkat želeli vstaviti v nalogi, smo uporabili elektroporacijo. Z optimizacijo postopkov smo opredelili uporabne pogoje za vstavljanje le-teh v človeške limfocite T. Limfocite T bi odvzeli pacientu, spremenili v laboratoriju in jih namnožili ter nato vrnili v periferno kri pacienta, kjer bi se izrazil protein konstrukta in opravil svojo nalogo. mRNA molekulskega konstrukta α-CAR-CD19 se je pri 1000 V ali 1100 V, dolžini pulza 40 ms izražala v 22 in 40,2 % celic celične linije Jurkat. S pravilno predpripravo in uspešno prepisanim molekulskim konstruktom v mRNA v procesu transkripcije in vitro, smo lahko zelo uspešni pri elektroporaciji vektorjev v celice pod danimi pogoji. Najpomembnejši postopek pri sami predpripravi pa je pravilna modifikacija mRNA. The biomedical utility of genetically modified immune cells (especially lymphocytes T) shows great value in treatment malignant diseases. Lymphocytes T can express various molecular constructs for therapeutic substances, that can influence the course of the disease. For the introduction of mRNA of molecular constructs of chimeric antigen receptors (CAR T), that we wanted to insert into the cells of cell lines Jurkat in the task, we used electrogenic transfer. By optimizing the procedures, we defined the useful conditions for inserting these into human lymphocites T. Lymphocytes T would be taken from the patient, changed in the laboratory and expanded, then returned to the patient's peripheral blood, where the protein of construct would be expressed and perform its task. The mRNA of the molecular construct α-CAR-CD19 was expressed in 22 and 40,2 % of cells of cell line Jurkat at 1000 V or 1100 V, a pulse length of 40 ms. With proper pre-preparation and successfully transcribed molecular construct into mRNA in the process of transcription in vitro, we can be very successful in electro gene transfer of vectors into cells under given conditions. The most important procedure in the preparation is the full modification of the mRNA.