A quantitative analysis of the dynamic of telomeres in human MJ-90hTERT cells

Abstract

Tema rada je kvantitativna analiza telomera u ljudskim stanicama MJ-90hTERT. To su fibroblasti imortalizirani telomerazom koja im omogućava produžavanje telomera, ali koji imaju fenotip i kontrolu rasta jednaku normalnim fibroblastima. Telomeraza produžuje pojedine telomere u ovisnosti o njihovoj konformaciji, odnosno duljini. Nakon označavanja telomera metodom hibridizacije in situ Q-PNA-FISH napravljena je kvantitativna analiza. Ovom se metodom može analizirati relativna duljina svake pojedine telomere i telomernog para na metafaznim kromosomima. Napravljena je usporedba duljina telomera sestrinskih kromatida fibroblasta koji su prošli 45, 60 i 75 udvostručenja populacije (PD) nakon unošenja telomeraze. Analiza promjena u duljini telomera pokazala je ujednačivanje njihove duljine razmjerno broju ciklusa kroz koji je stanična populacija prošla. Također je statistički pokazana pouzdanost metode Q-PNA-FISH u analizi telomera.The topic of this master thesis is a quantitative analysis of the dynamic of telomeres in human MJ-90 hTERT cells. This fibroblast cell line is immortalized by telomerase which enables telomere lenghtening, but the cells retain the same phenotype and growth control as in normal fibroblasts. Telomerase lenghtens telomeres in dependence on their conformation and/or length. Telomeres in these cells are quantitatively analyzed with software after the in situ hybridization with the Q-PNA-FISH method. This method enables analysis of each telomere and telomere pair on metaphase chromosomes. The length of sister chromatid telomeres were compared in fibroblasts which grew for 45, 60 and 75 population doublings in culture, after immortalization by telomerase. The analysis of changes in telomere length showed tendency of equalization in telomere length during the growth in the culture. Besides that, the reliability of Q-PNA-FISH in telomere analysis was statistically demonstrated