magistrsko delo
Tjaša Škerjanec (Author), Matej Butala (Reviewer), Maja Čemažar (Mentor), Maja Čemažar (Thesis defence commission member), Matej Butala (Thesis defence commission member), Tom Turk (Thesis defence commission member), Maša Bošnjak (Thesis defence commission member), Maša Bošnjak (Co-mentor)

Abstract

Vnašanje tujih nukleinskih kislin v celice z elektroporacijo za zdravljenje rakavih obolenj se trenutno uveljavlja kot vedno bolj učinkovita metoda zdravljenja raka v veterini in medicini. Učinkovitost genskega elektroprenosa je odvisna od fizikalnih dejavnikov električnega polja, vrste vstavljene nukleinske kisline in bioloških dejavnikov tkiva. Genski elektroprenos aktivira številne citosolne senzorje nukleinskih kislin, kar vodi v aktivacijo imunskega odziva z vnetnimi citokini. Želeli smo ugotoviti, ali je genski elektroprenos nukleinskih kislin citotoksičen in ali povzroči aktivacijo citosolnih senzorjev nukleinskih kislin v humanih celicah melanoma Sk Mel28 ter adenokarcinoma debelega črevesa in danke HT29. Opazovali smo tudi morfološke spremembe celic po genskem elektroprenosu. Ugotovili smo, da genski elektroprenos po protokolu EP2 na celice Sk Mel28 nima citotoksičnega učinka, pri celicah HT29 pa je citotoksičnost odvisna od fizikalnih lastnosti pulzov. Pri opazovanju morfoloških sprememb smo opazili, da po genskem elektroprenosu pri določenem deležu celic pride do apoptoze ali nekroze, tip celične smrti pa je odvisen od fizikalnih lastnosti pulzov. Pri celični liniji Sk Mel28 z genskim elektroprenosom nukleinskih kislin nismo zvišali izražanja genov citosolnih senzorjev nukleinskih kislin, pri HT29 pa smo z genskim elektroprenosom z nekaterimi nukleinskimi kislinami, predvsem s plazmidom Vax, neodvisno od fizikalnih lastnosti pulzov, povečali izražanje genov citosolnih senzorjev DDX60, RIG I in IFI16 ter vnetnega citokina dejavnik tumorske nekroze α.

Keywords

genski elektroprenos;elektroporacija;citosolni senzorji nukleinskih kislin;citotoksičnost;melanom Sk-Mel28;adenokarcinom HT29;qPCR;interferonski imunski odziv;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UP FVZ - College of Health Care Izola
Publisher: [T. Škerjanec]
UDC: 577.2(043.2)
COBISS: 5118799 Link will open in a new window
Views: 802
Downloads: 208
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Other data

Secondary language: English
Secondary title: ǂThe ǂeffect of gene electrotransfer on cytosolic sensors and cytokines in humane cells in vitro
Secondary abstract: Introduction of foreign nucleic acids into cells via electroporation with the intention of treating cancer diseases is becoming more and morerecognised as an effective treatment method in veterinary and human medicine. The efficiency of gene electrotransfer depends on the physical properties of the electrical field, the type of inserted nucleic acid and the biological tissue factors. The gene electrotransfer activates numerous cytosolic sensors for nucleic acids, which leads to the activation of a immune response with the help of inflammatory cytokines. We investigated whether the gene electrotransfer of nucleic acids is cytotoxic and whether it causes the activation of cytosolic sensors in the human Sk-Mel28 melanoma cells and the HT29 colorectal adenocarcinoma cells. We also observed the morphological changes of cells following gene electrotransfer. We conclude that gene electrotransfer to Sk-Mel28 cells, using EP2 protocol, has no cytotoxic effect, whereas in HT29 cells the cytotoxicity depends on the physical properties of the pulses. During observation of morphological changes, we noticed that after gene electrotransfer a certain percentage of cells became apoptotic or necrotic, and the type of cell death depended on the physical properties of the pulses. In the Sk-Mel28 cell line, we did not observe increased expression of cytosolic sensorsm after GET, whereas in HT29 after GET of selected nucleic acids, primarily the Vax plasmid, we observe increased expression of cytosolic sensors DDX60, RIG-I and IFI16 independently of the properties of the electroporation pulses. Increased expression of the inflammatory cytokine tumour necrosis factor α was also observed.
Secondary keywords: gene electrotransfer;electroporation;cytosolic sensors;cytotoxicity;melanoma cells Sk-Mel28;adenocarcinoma cells HT29;qPCR;interferon immune response;
Type (COBISS): Master's thesis/paper
Study programme: 0
Thesis comment: Univ. Ljubljana, Biotehniška fak.
Pages: XV f., 79, [1] str.
ID: 11187523