Priprava pasjih matičnih celic kot dostavni sistem interlevkina 12 za zdravljenje tumorjev.

Pirih, Nina (author), Majdič, Gregor (mentor), Kamenšek, Urška (comentor)

Mezenhimske matične celice zaradi svojega širokega potenciala diferenciacije v različne vrste celic in produkcije rastnih faktorjev in različnih komponent zunajceličnega matriksa, ponujajo številne možnosti uporabe v terapevtske namene. Poleg njihove uporabe pri regeneraciji tkiv in modulaciji imunskega sistema, lahko služijo tudi kot dostavni sistemi pri zdravljenju rakavih obolenj, saj raziskave kažejo, da mezenhimske matične celice pogosto migrirajo k tumorskim tkivom. Citokin interlevkin 12 (IL-12) zaradi svoje sposobnost aktivacije naravne in pridobljene imunost ter stimulacije produkcije citokina interferona ?, ki koordinira protitumorsko obrambo, predstavlja idealnega kandidata za tumorsko imunoterapijo. Cilj naloge je bil z ne-virusnimi metodami pridobiti linijo stabilno transfeciranih pasjih matičnih celic, ki bi v zadostni meri proizvajale IL-12, da bi lahko potencialno služile v namen protitumorske terapije pri psih. V nalogi smo uporabili metodo elektroporacije in lipofekcije. Z uporabo reporterskega plazmida pEGFP N1 smo optimizirali pogoje elektroporacije, ki smo jih kasneje uporabili pri elektroporacijah s terapevtskim plazmidom pcDNA3.1zeo+sccaIL12. V optimizacijo smo vključili elektroporacijske parametre, število celic/elektroporacijo, količino plazmidne DNA/elektroporacijo in vrsto elektroporacijskega pufra. Stabilne linije pasjih matičnih celic, ki bi izražale caIL-12, nam po večkratnih poskusih elektroporacije in selekcije ni uspelo pridobiti, vendar smo s qPCR metodo uspeli dokazati prehodno izražanje caIL-12 na mRNA nivoju. Rezultati magistrskega dela so postavili dobre temelje za transfekcijo pasjih matičnih celic pridobljenih iz maščobnega tkiva.

• Language: Slovenian
• Keywords: pasje matične celice, mezenhimske matične celice, interlevkin 12, IL-12, imunoterapija, rak, tumor, transfekcija, elektroporacija, genski elektroprenos, lipofekcija, dostavni sistem
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [N. Pirih]
• Year: 2019
• PID: 20.500.12556/RUL-111074
• UDC: 602.9:591.81:602.64(043.2)
• COBISS.SI-ID: 9345913
• Publication date in RUL: 22.09.2019

PIRIH, Nina, 2019, Priprava pasjih matičnih celic kot dostavni sistem interlevkina 12 za zdravljenje tumorjev. [online]. Master’s thesis. N. Pirih. [Accessed 26 March 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=111074

Secondary language

• Language: English
• Title: Preparation of canine stem cells expressing interleukin 12 as delivery system for tumor therapy

Abstract

Mesenchymal stem cells, due to their wide potential for differentiation into different cell types and the production of growth factors and various components of the extracellular matrix, offer many possibilities for therapeutic use. In addition to their use in tissue regeneration and in the modulation of the immune system, they can also serve as delivery systems for the treatment of cancer, as studies have shown that mesenchymal stem cells often migrate towards tumor tissue. Cytokine interleukin 12 (IL-12), due to its ability of activation natural and adaptive immunity and stimulation of the production of cytokine interferon ?, which coordinates anti-tumor defense, is an ideal candidate for tumor immunotherapy. The goal of our work was to obtain a stable transfected line of canine stem cells with non-viral methods, that adequately express IL-12 and could potentially be a part of anti-tumor therapy in dogs. We used the method of electroporation and lipofection. Using the reporter plasmid pEGFP N1, we optimized the conditions of electroporation, which were later used in electroporations with the therapeutic plasmid pcDNA3.1zeo + sccaIL12. The optimization included the electroporation parameters, the number of cells/electroporation, the amount of plasmid DNA/electroporation, and the type of electroporation buffer. We were not able to obtain stable transfected canine stem cells that express caIL-12 despite repeated attempts of electroporation and selection. However, with the qPCR method we successfully proved transient expression of caIL-12 at the mRNA level. The results of the master thesis have laid out good foundation for the transfection of canine stem cells derived from fat tissue.

• Keywords: canine stem cells, mesenchymal stem cells, interleukin 12, IL-12, immunotherapy, cancer, tumor, transfection, electroporation, gene electrotransfer, lipofection, delivery system