Korištenje adenovirusnih vektora u istraživanju ciklusa tumorskih stanica

Pranjić, Blanka

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master's thesis

Korištenje adenovirusnih vektora u istraživanju ciklusa tumorskih stanica

Zagreb: University of Zagreb, Faculty of Science, 2008. urn:nbn:hr:217:645812

Pranjić, Blanka

University of Zagreb
Faculty of Science
Department of Biology

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APA 6th Edition

Pranjić, B. (2008). Korištenje adenovirusnih vektora u istraživanju ciklusa tumorskih stanica (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:645812

MLA 8th Edition

Pranjić, Blanka. "Korištenje adenovirusnih vektora u istraživanju ciklusa tumorskih stanica." Master's thesis, University of Zagreb, Faculty of Science, 2008. https://urn.nsk.hr/urn:nbn:hr:217:645812

Chicago 17th Edition

Harvard

Pranjić, B. (2008). 'Korištenje adenovirusnih vektora u istraživanju ciklusa tumorskih stanica', Master's thesis, University of Zagreb, Faculty of Science, accessed 25 November 2024, https://urn.nsk.hr/urn:nbn:hr:217:645812

Vancouver

Pranjić B. Korištenje adenovirusnih vektora u istraživanju ciklusa tumorskih stanica [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2008 [cited 2024 November 25] Available at: https://urn.nsk.hr/urn:nbn:hr:217:645812

IEEE

B. Pranjić, "Korištenje adenovirusnih vektora u istraživanju ciklusa tumorskih stanica", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2008. Available at: https://urn.nsk.hr/urn:nbn:hr:217:645812

Cite this item: https://urn.nsk.hr/urn:nbn:hr:217:645812

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Metadata

Title	Korištenje adenovirusnih vektora u istraživanju ciklusa tumorskih stanica
Title (english)	Cell cycle analysis of tumor cells using adenoviral vectors
Author	Blanka Pranjić
Mentor	Marijeta Kralj (mentor)
Mentor	Maja Matulić (mentor)
Committee member	Maja Matulić (predsjednik povjerenstva)
Committee member	Marijeta Kralj (član povjerenstva)
Committee member	Nada Oršolić (član povjerenstva)
Committee member	Željka Vidaković Cifrek (član povjerenstva)
Granter	University of Zagreb Faculty of Science (Department of Biology) Zagreb
Defense date and country	2008, Croatia
Scientific / art field, discipline and subdiscipline	NATURAL SCIENCES Biology
Abstract	Poremećaj regulacije staničnog ciklusa uzrokuje nekontroliranu proliferaciju stanica te je jedan od glavnih uzroka pojave raka. Postoji niz gena koji reguliraju stanični ciklus i čuvaju stanice od zloćudne preobrazbe, među kojima je gen p21WAF1/CIP1 čiji proteinski produkt je inhibitor kompleksa kinaze ovisne o ciklinu (CDK) i ciklina. Njegova povećana ekspresija u tumorskim stanicama predstavlja potencijalno oružje u genskom liječenju raka, međutim istraživanje njegove uloge ukazuje na niz kontradiktornih rezultata zbog kojih je nužno dodatno rasvijetliti njegovu ulogu u tumorskim stanicama. U ovom je radu praćen utjecaj povećane ekspresije gena p21 unošenjem njegove cDNA replikacijski nesposobnim adenovirusnim vektorima (Ad5-p21) u tumorske stanice karcinoma debelog crijeva SW480 i SW620, koje potječu od istog pacijenta, ali imaju različit stupanj zloćudne preobrazbe. Kao kontrolni vektor je korišten adenovirus s ugrađenim genom dojavljivačem (Ad5-β-gal). Oba vektora umnožena su u visokoj koncentraciji u komplementirajućim stanicama HEK 293, izolirana i pročišćena. Virusni titar je izmjeren određivanjem koncentracije ukupnih i infektivnih virusnih čestica. Djelotvornost transdukcije stanica karcinoma debelog crijeva SW620, SW480, a i HCT116 adenovirusnim vektorom Ad5-β- gal određena je praćenjem pojave plavog obojenja aktivnošću enzima β-galaktozidaze. Uočena je slabija djelotvornost transdukcije SW620 stanica putem Ad5-β-gal u odnosu na SW480 i HCT116 stanice. Također je primijećeno toksično djelovanje samog vektora na tumorske stanice, poglavito na stanice SW620. Utjecaj Ad5-p21 i Ad5-β-gal na stanični ciklus SW620 i SW480 stanica praćen je metodom protočne citometrije. Povećana ekspresija gena p21 izaziva zastoj u G1 fazi staničnog ciklusa stanica SW480. Njegova je uloga u stanicama SW620 upitna zbog snažnog utjecaja samog adenovirusnog vektora na stanični ciklus, budući da je uočen zastoj staničnog ciklusa u G2 fazi nakon zaražavanja stanica s kontrolnim vektorom Ad5-β-gal. Kako i sami adenovirusni vektori mogu utjecati na stanični ciklus te uzrokovati lažne rezultate, potreban je izuzetan oprez kod njihovog korištenja u istraživanju gena koji reguliraju stanični ciklus.
Abstract (english)	Deregulation of the cell cycle induces uncontrolled cell proliferation and is one of the main causes of cancer development. A variety of genes has cell cycle regulating properties and can therefore protect cells from malignant transformation. An example is p21WAF1/CIP1 which is an inhibitor of cyclin-dependent kinases (CDK)/cyclin complexes. Overexpression of p21WAF1/CIP1 in tumor cells might represent a potential tool for cancer gene therapy. However, the study of p21WAF1/CIP1 functions in tumor cells revealed contradictory findings and therefore it is necessary to clarify its role in tumor cells with different genetic background. The effect of p21WAF1/CIP1 overexpression was monitored by introducing p21 cDNA into colon carcinoma cells SW480 and SW620, using the replication-deficient recombinant adenoviral vectors. These cell lines were derived from the same patient, but from cells with different level of malignancy. Adenoviral vector containing the reporter gene for β-galactosidase was used as a control. Both vectors were propagated in HEK 293 helper cells in high concentrations, isolated and purified. Viral titer was determined measuring the concentration of total and infectious viral particles. The efficiency of transduction of colon carcinoma cells SW620, SW480, as well as HCT116, with adenoviral vectors was determined by β-galactosidase staining of infected cells. Transduction of SW620 with Ad5-β-gal had lower efficiency, as compared to SW480 and HCT116. In addition, a vector related toxic effect on tumor cells was detected, in particular on SW620 cells. The effect of Ad5- p21 and Ad5-β-gal on the cell cycle of SW620 and SW480 cells was monitored by flow cytometry. p21WAF1/CIP1 overexpression in SW480 cells induced cell cycle arrest in G1 phase. In SW620, a strong effect of the vector itself, Ad5-βgal, was detected, resulting in G2/M arrest. Since the adenoviral vector itself can effect the cell cycle leading to false results, their use in the investigation of the cell cycle regulating genes should be handled with care.
Keywords
Keywords (english)
Language	croatian
URN:NBN	urn:nbn:hr:217:645812
Study programme	Title: Molecular Biology Study programme type: university Study level: graduate Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
Type of resource	Text
File origin	Born digital
Access conditions	Open access
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Repository	Repository of the Faculty of Science
Created on	2018-03-05 12:24:25