diplomsko delo
Klara Ažbe (Avtor), Katarina Černe (Mentor)

Povzetek

Rak jajčnikov je eden izmed najpogostejših ginekoloških rakov, ki zaradi poznega odkritja pogosto vodi v smrt. Zdravljenje je v napredovalih stadijih velikokrat neuspešno zaradi odpornosti na zdravila na osnovi platine (Pt-BM). Potencialni biooznačevalec za napoved odpornosti na Pt-BM je ATP7A. Na podlagi določanja ATP7A bi lahko izboljšali zdravljenje in preživetje bolnic z rakom jajčnikov. Vendar bi morali pomen določanja izražanja ATP7A dodatno ovrednotiti. Namen diplomske naloge je bil optimizacija protokola za določanje izražanja ATP7A s pretočno citometrijo, ki je občutljiva in zmogljiva metoda. Do sedaj so namreč izražanje ATP7A v celicah raka jajčnikov določali z drugimi metodami, kot so prenos western, qPCR in konfokalna mikroskopija. Ker se prenašalec nahaja predvsem znotraj celice, je za njegovo detekcijo potrebno izvesti intracelularno barvanje, ki pa je časovno in tehnično zahtevno. Zanimalo nas je, če pride do razlik v nivoju izražanja ATP7A med celicami, ki jih najprej gojimo in zamrznjene shranimo, nato pa pred poskusom odmrznemo (v nadaljevanju »zamrznjene celice«) in celicami, ki jih najprej gojimo v celični kulturi in potem, ko jih odlepimo, takoj uporabimo za poskus (v nadaljevanju »rastoče celice«). Poskuse smo izvedli na celični liniji PEO1, ki je bila izolirana iz bolnice z napredovalim rakom jajčnikov. Izvedli smo dva tipa poskusov, tj. na zamrznjenih in rastočih celicah. Ugotovili smo, da predhodna zamrznitev vpliva na proces permeabilizacije in sicer tako, da pri zamrznitvi najverjetneje pride do poškodb membrane in zato hitreje pride do naluknjanja le-te. Med pomembnejše karakteristike celic, ki vplivajo na poskus, spada viabilnost celic, ki mora presegati 90 %. Pomemben dejavnik, ki vpliva na rezultate, je izvajalec poskusa. Določali smo tudi optimalno napetost na citometru, od katerega je odvisna jakost signala. Ugotovili smo, da je bila izhodiščna napetost citometra optimalna. Na osnovi povprečne intenzitete fluorescence in odstotka fluorescentnih dogodkov smo prišli do zaključkov, da na rezultate znotrajcelične ekspresije ATP7A ne vpliva, če analizo s pretočno citometrijo izvedemo na zamrznjenih celicah. Ponovljivost rezultatov znotrajcelične ekspresije ATP7A se je med poskusi in znotraj poskusov na rastočih in zamrznjenih celicah posebej, izkazala za sprejemljivo. Na podlagi pridobljenih rezultatov lahko sklepamo, da bi lahko v prihodnje izvajali poskuse tako na zamrznjenih kot na rastočih celicah. Optimalno bi bilo narediti štiri paralelke vzorcev znotraj poskusa in poskus štirikrat ponoviti.

Ključne besede

odpornost na zdravljenje;bakrovi transporterji;ATPaze;ATP7A;pretočna citometrija;optimizacija;diplomska dela;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.11 - Diplomsko delo
Organizacija: UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik: [K. Ažbe]
UDK: 576(043.2)
COBISS: 170025987 Povezava se bo odprla v novem oknu
Št. ogledov: 74
Št. prenosov: 8
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Optimization of the protocol for the determination of ATP7A in an ovarian cancer cell line by flow cytometry.
Sekundarni povzetek: Ovarian cancer is one of the most common gynecological cancers, which often leads to death due to late detection. Treatment is often unsuccessful in advanced stages due to resistance to platinum-based drugs (Pt-BM). A potential biomarker to predict Pt-BM resistance is ATP7A. Based on the determination of ATP7A, the treatment and survival of patients with ovarian cancer could be improved. However, the significance of determining ATP7A expression should be further evaluated. The aim of the thesis was to optimize the protocol for determining the expression of ATP7A by flow cytometry, which is a sensitive and powerful method. Until now, the expression of ATP7A in ovarian cancer cells has been determined by other methods, such as western blotting, qPCR and confocal microscopy. Since the transporter is mainly located inside the cell, for its detection it is necessary to carry out intracellular staining, which is time-consuming and technically demanding. We were interested in whether there are differences in the level of ATP7A expression between cells that are first grown and stored frozen and then thawed before the experiment (hereafter "frozen cells") and cells that are first grown in cell culture and after peel them off, immediately use them for the experiment (hereinafter "growing cells"). The experiments were performed on the PEO1 cell line, which was isolated from a patient with advanced ovarian cancer. We performed two types of experiments, i.e. on frozen and growing cells. We found that prior freezing affects the permeabilization process in such a way that, during freezing, the membrane is most likely to be damaged and, therefore, perforation of the membrane occurs more quickly. Among the more important cell characteristics that affect the experiment is cell viability, which must exceed 90 %. An important factor influencing the results is also the experimenter. We also determined the optimal voltage on the cytometer, on which the signal strength depends. We found that the baseline voltage of the cytometer was optimal. Based on the average fluorescence intensity and the percentage of fluorescent events, we came to the conclusions that the results of the intracellular expression of ATP7A are not affected if the flow cytometry analysis is performed on frozen cells. The reproducibility of intracellular ATP7A expression results between and within experiments on growing and frozen cells separately was found to be acceptable. Based on the obtained results, we can conclude that in the future experiments could be performed on both frozen and growing cells. It would be optimal to make four parallel samples within the experiment and repeat the experiment four times.
Sekundarne ključne besede: ovarian cancer;resistance;ATP7A;flow cytometry;optimization;Novotvorbe jajčnikov;Univerzitetna in visokošolska dela;
Vrsta dela (COBISS): Diplomsko delo/naloga
Študijski program: 1000371
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Strani: 58 str.
ID: 19909059