20.500.12556/RUL-129574 Vloga proteina MPP7 pri diferenciaciji celične linije človeških osteoblastov MG63 The role of MPP7 protein in human osteoblast cell line MG63 differentiation Vsegenomske študije (GWAS; angl. Genome Wide Association Study) imajo pomembno vlogo pri iskanju novih terapevtskih tarč. S primerjavo pojavnosti genetskih variant pri zdravih in obolelih nam ta način preiskovanja omogoča lokalizacijo mest v genomu in prepoznavo genov (posredno tudi proteinov), ki vplivajo na opazovano lastnost. S pomočjo te metode so ugotovili, da obstaja močna povezava med kostno gostoto in variantami membranskega palmitoiranega proteina 7 (MPP7; angl. Membrane Palmitoylated Protein 7). Kljub temu pa še vedno ni povsem znano, kakšna je njegova vloga pri diferenciaciji in aktivaciji kostnih celic. V sklopu magistrske naloge smo želeli potrditi vlogo proteina MPP7 pri diferenciaciji osteoblastov. Kot celični model smo uporabili celice osteosarkoma MG-63. Primerjali smo stopnjo diferenciacije med celicami z izbitim genom za protein MPP7 (MMP7 KO) in kontrolnimi nespremenjenimi celicami MG-63, ki imajo ohranjeno sposobnost izdelave omenjenega proteina. Pri ocenjevanju stopnje razvitosti osteoblastov smo si pomagali z več različnimi metodami. Stopnjo razvoja smo ocenjevali na podlagi izražanja kazalnih genov, značilnih za diferenciacijo osteoblastov, pri čemer smo uporabili metodo kvantitative verižne reakcije s polimerazo (RT-qPCR). Določili smo izražanje gena za z RUNT povezanim transkripcijskim dejavnikom 2 (RUNX2; angl. Runt-Related Transcription Factor 2), gena za alkalno fosfatazo (ALP; angl. Alkaline Phosphatase), gena za kolagen tipa I alfa 1 verige (COL1A1; angl. Collagen Type I Alpha 1 Chain ) in gena za kostni gama karboksglutamat protein oziroma osteokalcin (BGLAP oz. OC; angl. Bone Gamma-Carboxyglutamate Protein). Izražanje posameznih kazalcev smo nato normalizirali glede na izražanje referenčnega gena za TATA-vezavni protein (TBP, angl. TATA-Box Binding Protein). Določili smo tudi kako se s potekom razvoja osteoblastov spreminja aktivnost encima ALP v celicah in celokupno koncentracijo proteinov v celičnih lizatih v različnih časovnih točkah razvoja. Rezultati so pokazali, da je bilo izražanje genov RUNX2 in COL1A1 značilno višje v MMP7 KO in izražanje gena ALP značilno nižje kot v kontrolnih celicah MG-63. Izražanje gena OC se je bilo obeh celičnih kulturah podobno. Prav tako smo opazili manjšo aktivnost ALP pri celicah z izbitim genom za protein MPP7. Med celičnima linijama ni bilo opaznih razlik v celokupni količini proteinov. Glede na pridobljene rezultate lahko sklepamo, da ima protein MPP7 vpliv na razvoj osteoblastov, saj je v celicah osteosarkoma MG-63 MPP7 KO prišlo do počasnejše diferenciacije celic, kot pri celicah, ki so izražale gen MPP7 (KC). Da bi ugotovili natančen mehanizem, preko katerega je protein MPP7 vpleten v celično diferenciacijo, bo potrebno izvesti dodatne raziskave funkcijske genomike. Genome wide association studies (GWAS) play a crucial role in discovery of potential novel therapeutic targets. By analysing the differences in occurrence frequency of genetic variants between healthy and diseased patients, the method enables us to find the locations in the genome, which influence the observed trait. With this method, variants in Membrane Palmitoylated Protein 7 (MPP7) were associated with bone mineral density. However, its role in regulation of bone mineral density is not determined yet. The purpose of this master thesis was to confirm that MPP7 protein is involved in differentiation of osteosarcoma cells MG-63, that we used as model cell line for osteoblasts. We compared two cell lines; knock-out MG-63 cells, which did not express a functional MPP7 protein (MPP7 KO), and control MG-63 cell line which still had the ability to produce MPP7 protein (KC). We used multiple methods to estimate the stage of osteoblast differentiation. The differentiation of both cell types was assessed using RT-qPCR method (reverse transcription quatitative Polymerase Chain Reaction) at different differentiation time points. We determined the expression of Runt-Related Transcription Factor 2 (RUNX2), Alkaline Phosphatase (ALP), Collagen Type I Alpha 1 Chain 1 (COL1A1) and Bone Gamma-Carboxyglutamate Protein (BGLAP or OC). Expression of each gene was normalized according to expression of reference gene, which was in our case gene for TATA-Box Binding Protein (TBP). We also measured Alkaline Phosphatase (ALP) activity and total protein content. Our results showed that RUNX2 and COL1A1 genes expression were higher in MPP7 KO cells than in control cells, but ALP gene expression was lower in MPP7 KO cells than in control cells. Expression of OC gene was similar in both cell cultures. We also found out that knock-out cells have lower ALP activity. But there were no differences between cell lines in total protein content. Based on our results we can conclude that MPP7 protein has some impact on differentiation of osteoblasts, because knock-out cells differentiated slower than cells expressing MPP7. More studies are needed to explore the exact mechanism, through which MPP7 is involved in cell differentiation. Osteoporoza osteoblasti diferenciacija membranski palmitoirani protein 7 (MPP7) alkalna fosfataza (ALP) Osteoporosis Osteoblasts Differentiacion Membrane Palmitoylated Protein 7 (MPP7) Alkaline Phosphatase (ALP). true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2021-09-05 07:45:16 2021-09-05 07:45:21 2022-09-08 04:09:22 0000-00-00 00:00:00 2021 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2022-09-01 83115 2697.pdf 2697.pdf 1 92786E0528FDDBE21B3277D4E651A0A7 77724f08d90ea0232939b7b4e943ed9eb80a96af4508c4828b9be98060cfe224 3a0f1c36-0e0c-11ec-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=146583 Fakulteta za farmacijo 0 0 0