Izvorni znanstveni članak
https://doi.org/10.3325/cmj.2013.54.489
Epigenetic drug 5-azacytidine impairs proliferation of rat limb buds in an organotypic modelsystem in vitro
Vedrana Mužić
; Department of Rehabilitation and Orthopaedic Devices, University Hospital Centre Zagreb, Zagreb Croatia
Ana Katušić Bojanac
; Department of Medical Biology, University of Zagreb, School of Medicine, Zagreb, Croatia
Gordana Jurić-Lekić
; Department of Histology and Embryology, University of Zagreb, School of Medicine, Zagreb, Croatia
Marta Himelreich
; Department of Histology and Embryology, University of Zagreb, School of Medicine, Zagreb, Croatia
Katarina Tupek
; Department of Medical Biology, University of Zagreb, School of Medicine, Zagreb, Croatia
Ljiljana Šerman
; Department of Medical Biology, University of Zagreb, School of Medicine, Zagreb, Croatia
Nina Marn
; Department of Medical Biology, University of Zagreb, School of Medicine, Zagreb, Croatia
Nino Sinčić
; Department of Medical Biology, University of Zagreb, School of Medicine, Zagreb, Croatia
Maja Vlahović
; Department of Medical Biology, University of Zagreb, School of Medicine, Zagreb, Croatia
Floriana Bulić-Jakuš
; Department of Medical Biology, University of Zagreb, School of Medicine, Zagreb, Croatia
Sažetak
Aim To establish an organotypic in vitro model of limb
bud development to verify whether epigenetic drug and
teratogen 5-azacytidine (5azaC) has an effect on limb buds
independent of its effects on the placenta.
Methods Fischer strain rat fore- and hindlimb buds were
microsurgically isolated from 13 days old embryos and cultivated
in vitro for two weeks at the air-liquid interface in
Eagle’s minimum essential medium (MEM) with 50% rat
serum. 30 μmol of 5azaC was added to the fresh medium.
Overall growth was measured by an ocular micrometer.
Routine histology, immunohistochemical detection of the
proliferating cell nuclear antigen (PCNA), and stereological
quantification of PCNA expression were performed.
Results At four time points, significantly lower overall
growth was detected for fore- and hindlimb bud explants
cultivated with 5azaC in comparison to controls. After
the culture period, numerical density of the PCNA signal
for both types of limb buds was lower than for controls
(P < 0.001). Limb buds were initially covered by immature
epithelium and contained mesenchyme, myotubes, single
hemangioblasts, hemangioblast aggregates, blood islands,
and capillaries. Regardless of the treatment, cartilage and
epidermis differentiated, but cells and structures typical for
vasculogenesis disappeared.
Conclusion Our findings, obtained outside of the maternal
organism, stress the importance of compromised cell
proliferation for 5azaC impact on limb buds. This investigation
points to the necessity to establish alternatives to in
vivo research on animals using teratogenic agents.
Ključne riječi
Hrčak ID:
117015
URI
Datum izdavanja:
15.10.2013.
Posjeta: 1.470 *