diplomsko delo
Nika Bedrač (Author), Katarina Černe (Mentor)

Abstract

Rak jajčnikov je najbolj smrtonosen ginekološki rak na svetu in osmi najpogostejši rak v Sloveniji. Težavno je že odkrivanje raka v zgodnjih fazah, saj se simptomi pojavijo le v poznem stadiju, takrat pa je zdravljenje precej zahtevno. Najpogosteje se uporabljajo zdravila na osnovi platine, med katerimi je zdravilo prvega izbora karboplatin. Tarča učinkovine je jedrna DNA, pri kateri pride do odvijanja in upognitve, s tem pa sta onemogočeni replikacija in transkripcija. Karboplatin velja za učinkovino z relativno malo resnimi neželenimi učinki, kar je tudi poglavitni razlog zakaj je nadomestil predhodno uporabljeno zdravilo cisplatin. Enega ključnih problemov pa predstavlja rezistenca na kemoterapevtike, saj se ta pojavi med zdravljenjem pri 80 % bolnic. Biooznačevalca za klinično napoved rezistence na zdravila na osnovi platine še ni na razpolago. ATP7A je proteaza tipa P in spada v skupino bakrovih transporterjev (eksporterjev) in skrbi za prenos bakra skozi membranski dvosloj ob porabljanju energije, ki izvira iz hidrolize ATP. Poleg bakra pa se na ATP7A lahko vežejo tudi spojine s platino. Zato je ATP7A obetajoč biooznačevalec pri ugotavljanju rezistence na karboplatin, ki pa ga je potrebno še ustrezno ovrednotiti. Do sedaj so v raziskavah ugotovili, da so opazovane celice, ki so bile rezistentne na omenjene kemoterapevtike, imele povišano izražanje ATP7A. Zaradi molekulske heterogenosti bolezni in ker so dosedanje raziskave izvedli samo na celicah neseroznega tipa raka jajčnikov, je prvi korak, da ugotovimo prisotnost ATP7A v celicah seroznega tipa raka jajčnikov. V okviru diplomskega dela smo želeli preveriti prisotnost ATP7A pri treh komercialno dostopnih celičnih linijah seroznega tipa raka jajčnikov: PEO14, OAW28 in OAW42. Celice smo najprej gojili v optimalnih pogojih, nato pa z imunološkim označevanjem dokazali prisotnost tarčnega biooznačevalca s tehniko pretočne citometrije. Za vse tri celične linije je značilno, da se med seboj zlepljajo v skupke, kar moti analizo s pretočno citometrijo. To smo želeli preprečiti z uporabo EDTA, DPBS in injekcijske brizge z iglama dveh velikosti. V naši raziskavi smo kot prvi dokazali znotrajcelično prisotnost ATP7A pri skoraj 90 % celic OAW28. Prvi smo ugotovili tudi odsotnost ATP7A na membrani celic OAW42. Največjo uspešnost pri ločevanju celic smo dosegli pri uporabi injekcijske brizge, vendar se je pri tem zmanjšala viabilnost. Med gojenjem celičnih linij smo naleteli na določene težave (okužba celic, slabo pritrjanje in nizko število celic), zaradi katerih nismo izvedli vseh načrtovanih meritev, predvsem ni uspela analiza PEO14. Ker smo upoštevali vsa navodila dobavitelja, je verjeten razlog zahtevnost gojenja PEO14, predvsem v smislu slabega pritrjanja na podlago, česar pa proizvajalec ni navedel.

Keywords

zdravila na osnovi platine;rezistenca;bakrovi transporterji;ATP7A;pretočna citometrija;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [N. Bedrač]
UDC: 577.2:618.11-006(043.2)
COBISS: 125679619 Link will open in a new window
Views: 48
Downloads: 8
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Other data

Secondary language: English
Secondary title: ATP7A as a potential biomarker for predicting carboplatin resistance in serous ovarian cancer
Secondary abstract: Ovarian cancer has globally the highest mortality rate among all gynecologic cancers world and is the eighth most common cancer type in Slovenia. Its major problem is discovering cancer in the early stages because symptoms typically show up only when it is already late for treatment. The most used drugs are chemotherapeutics based on platinum, with carboplatin being the most important. Nuclear DNA is the main target, which breaks and bends so that replication and transcription are not possible. Carboplatin has a relatively low toxicity, which is the reason why it replaced other platinum-based drugs. The key problem of ovarian cancer chemotherapy is resistance to platinum-based drugs, which occurs in 80 % of the patients. ATP7A is a P-type protease and is a member of Cu-transporters (exporters) that are important for exporting copper from cells through the cell membrane. They utilize energy from ATP hydrolysis. In addition to copper, they can also bind platinum. That is why ATP7A is an important potential biomarker for predicting resistance to platinum-based medicine, but it still needs to be verified. Previous research shows that cells resistant to platinum-based treatment have a higher expression rate of ATP7A. Because of the molecular heterogeneity of the disease, research is mostly made on non-serous cell lines and that is the reason why determining the presence of ATP7A in serous cell lines remains the first next important step. In this work, we wanted to prove the presence or absence of ATP7A in commercially available cell lines PEO14, OAW28 and OAW42. We cultivated cells in optimal growth conditions and with immunostaining, we established the presence of ATP7A using flow cytometry. Because all three cell lines are prone to cell aggregation, which could interrupt our analysis, we also wanted to overcome this problem by using EDTA, DPBS, and an injection syringe. In this work, we were the first to prove the intracellular localization of ATP7A in almost 90 % of the OAW28 cells. On top of that, we were the first to prove the extracellular absence of ATP7A in OAW42. The best approach for solving the problem of cells aggregation was the usage of an injection syringe, but that resulted in lower viability of the cells. During cell activation, we encountered some problems, such as infection, poor adhesion, and low cell density. This is the reason why not all parameters are shown in this work. The most problematic cell line was PEO14. We followed all the instructions provided by the supplier of the cell line, but inability of these cells to stick to flask plastics was not mentioned.
Secondary keywords: ovarian cancer;resistance;ATP7A;flow cytometry;Novotvorbe jajčnikov;Univerzitetna in visokošolska dela;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 39 str.
ID: 16372692