20.500.12556/RUL-145057 Vzpostavitev in molekularna opredelitev celične linije mišjega oralnega ploščatoceličnega karcinoma MOC1 s stabilnim izražanjem onkoproteinov E6 in E7 Preparation and molecular characterization of cell line of murine oral squamous cell carcinoma MOC1 with stable expression of oncoproteins E6 and E7 Ploščatocelični karcinomi glave in vratu (PCKGV) so pogosta rakava obolenja z visoko umrljivostjo, ki jih lahko povzroči tudi humani papiloma virus (HPV). Okužbe s HPV lahko pri PCKGV razdelimo na produktivno in neproduktivno okužbo. Pri neproduktivni okužbi pride do vključitve virusnega genoma v gostiteljski genom, kar zaradi nenadzorovanega izražanja onkoproteinov E6 in E7 pripelje do maligne transformacije celic. Proteina E6 in E7 vplivata na protein 53 (p53) in protein retinoblastoma (pRB), ki sta odgovorna za uravnavanje celičnega cikla ter sprožitev apoptoze. V nalogi smo pripravili dve HPV-pozitivni celični liniji mišjega PCKGV MOC1 z virusno transdukcijo genov za proteina E6 in E7 ter določili uspešnost priprave celične linije glede na izražanje onkoproteinov E6 in E7 na nivoju mRNA in proteinov. Iz celičnih linij smo izolirali RNA, ki smo ji določili koncentracijo in čistost, nato pa smo jo uporabili za analizo relativnega izražanja genov za onkoproteina E6 in E7 glede na hišna gena za beta-aktin in gliceraldehid 3-fosfat dehidrogenazo (GAPDH). Pripravili smo tudi celične preparate, ki smo jih imunofluorescenčno pobarvali s protitelesi proti onkoproteinoma E6 in E7 ter tako preverili izražanje omenjenih onkoproteinov na ravni proteinov. Dokazali smo, da je izražanje stabilno na obeh nivojih. Head and neck squamous cell carcinomas (HNSCCs) are common cancers with high mortality, that can be caused by human papilloma virus (HPV). In HNSCC, HPV infections can be divided into productive and non-productive infection. In non-productive infection, the viral genome is incorporated into the host genome, which leads to malignant transformation of cells due to the uncontrolled expression of oncoproteins E6 and E7. Oncoproteins E6 and E7 affect the proteins p53 and pRB, which are responsible for regulating the cell cycle and triggering apoptosis. We prepared HPV-positive murine HNSCC MOC1 cell line by viral transduction of genes for oncoproteins E6 and E7, and evaluated the established cell lines, based on expression of proteins E6 and E7 at mRNA and protein levels. RNA was isolated from the cell lines, it's concentration and purity determined, and then RNA was used to analyze the expression of E6 and E7 proteins, relative to the housekeeping genes for beta-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We also prepared fixed cell samples, which were immunofluorescently stained with antibodies against proteins E6 and E7, thus determining expression of E6 and E7 at the protein level. We have demonstrated that expression across passages was stable at both levels. ploščatocelični karcinom glave in vratu humani papiloma virus MOC1 E6 E7 pRB p53 radioterapija ARD retrovirusna transdukcija head and neck squamous cell carcinoma HPV MOC1 E6 E7 pRB p53 radiotherapy ARD retroviral transduction true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2023-04-01 07:15:21 2023-04-01 07:15:27 2023-10-06 12:22:50 0000-00-00 00:00:00 2023 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 217609 147559427 32083.pdf 32083.pdf 1 B086C7E9EE100CF071706AB0EFE1DBF7 b84fb375ce034e8db101d07558fd9895a6edaf3225772ee5b055f72225b0a26f 1a5aef81-d04c-11ed-a314-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=167686 Biotehniška fakulteta 0 0 0