Testiranje izbranih učinkovin za inhibicijo modificirane 6-fosfofrukto-1-kinaze v metastaznih rakastih celicah.

Čamernik, Katja (avtor), Legiša, Matic (mentor)

Encim 6-fosfofrukto-1-kinaza je eden od najpomembnejših regulatornih encimov glikolize. Pri ljudeh najdemo 3 tipe encima, ki so različno izraženi v posameznih tkivih. Za številne tipe raka je značilna, povečana ekspresija izoforme Pfk-L, ki smo jo v raziskavi uporabili, za testiranje inhibicije aktivnosti, ob dodatku izbranih inhibitorjev. Z Gibson assembly tehnologijo smo v plazmidnem vektorju p416GPD pripravili rekombinantni humani nPFKL in sfPFKL gen in ju vnesli v PFK-null sev kvasovke (HD114-8D). Encim nPfk-L smo po namnožitvi v kvasovkah izolirali do homogenosti s frakcionirnim obarjanjem in afinitetno kromatografijo, medtem, ko smo zaradi nestabilnosti transformante sfPFKL, encim sfPfk-L le delno prečistili s frakcionirnim obarjanjem z amnosulfatom. Oba očiščena encima smo uporabili za testiranje inhibicije aktivnosti ob dodatku 15 izbranih inhibitorjev. Uspeli smo določiti tiste, ki bolje inhibirajo aktivnost nPfk-L encima, oziroma tiste, ki bolje inhibirajo sfPfk-L encim, kar predstavlja potencial za tarčno zdravljenje tumorjev. Ugotovili smo tudi, da so transformante z vnesenim nPFKL precej stabilne medtem, ko je sam encim nPfk-L v 30 min neprekinjene meritve, praktično izgubil vso aktivnost. Obratno pa so transformante z vnesenim sfPFKL genom izredno nestabilne, saj so precej hitro izgubile plazmid z vnesenim genom, sam encim sfPfk-L, ki smo ga nekaj sicer uspeli izolirati iz sveže pripravljene transformante pa je bil bolj stabilen.

• Jezik: Slovenski jezik
• Ključne besede: metabolizem rakavih celic, rakave celice, encimi, laktat, Pfk-L encim, NADH
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: BF - Biotehniška fakulteta
• Založnik: [K. Čamernik]
• Leto izida: 2018
• PID: 20.500.12556/RUL-103957
• UDK: 606:616-006.6:577.15(043.2)
• COBISS.SI-ID: 9047929
• Datum objave v RUL: 29.09.2018

ČAMERNIK, Katja, 2018, Testiranje izbranih učinkovin za inhibicijo modificirane 6-fosfofrukto-1-kinaze v metastaznih rakastih celicah. [na spletu]. Magistrsko delo. K. Čamernik. [Dostopano 16 april 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=103957

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Testing selected substances for inhibition of modified 6-phosphofructo-1-kinase in metastatic cancer cells

Izvleček

Enzyme 6-phosphofructo-1-kinase is one of the most important regulatory enzymes of glycolysis. There are three phosphofructokinase genes in humans, that are diversely expressed in specific human tissue types. Enchanced expression of Pfk-L isoform, that we used for testing of inhibition upon addition of selected inhibitors, is characteristic for many human tumor types. We used Gibson assembly technology and p416GPD plasmid vector to prepare human nPFKL and sfPFKL gene, and inserted them into PFK-null yeast strain (HD114-8D). Enzyme nPfk-L was purified to homogeneity with ammonium sulfate fractional precipitation and affinity chromatography, while we purified sfPfk-L only with fractional precipitation, due to very low level of extracted sfPfk-L protein, from unstable sfPFKL yeast strain. We used both purified enzymes to test inhibition of activity, upon addition of 15 seleted inhibiotrs. We were able to determine those inhibitors, that show better inhibiton for nPfk-L activity, or sfPfk-L activity respectively. We also noticed that transformants with inserted nPFKL were quite stable, while the enzyme nPfk-L practically lost all of it`s activity after 30 min of continuous measurement. On the contrary, transformants with inserted sfPFKL were very unstable, they lost the majority of their inserted plasmids, while sfPfk-L enzyme preserved its activity.

• Ključne besede: metabolism of cancer cells, cancer cells, enzyme, lactate, Pfk-L enzyme, NADH