hrvatski

Regulation of growth and differentiation in early mammalian embryo by growth factors FGF and NGF in organ culture in vitro

The aim was to analyse regulation of growth and tissue differentiation in a unique in vitro model of gastrulating mammalian embryo by fibroblast growth factor (FGF) and nerve growth factor (NGF) during two weeks. They both play a crucial role during embryogenesis and purpose of current study was to test their possible synergistic influence in the period when mammalian embryos are extremly sensitive on external factors. We cultivated 9, 5-days-old rat embryos on a metal grid supported lens paper, at air-liquid interface in culture medium (Eagle's minimal essential medium (MEM) with 50% of homologuos serum) with addition of FGF, NGF and combination of FGF and NGF in time frame of 9 days. Other 3 groups of embryos were 24 hours pretreated with 5-azacytidine (5-azaC), agent which can activate repressed genes. Parallel group of non-treated control embryos were cultivated with every experimental group. During 14 days of culture embryos grown in teratoma-like explants, and growth rate was evaluated by measuring average size of explants using an eyepiece micrometer on a few particular days (5, 7, 11, 14), after addition of growth factors. Differences between respected groups were estimated by Student's t-test.Differentiated tissues were estimated on serial histological sections. Chi-square test or Fischer exact test were used to compare the proportion of tissues between respected groups. In embryo derived teratomas NGF, and FGF/NGF combination used in time frame of 9 days did not stimulate differentiation of any kind of tissues, moreover FGF/NGF inhibited maturation of epidermis, while FGF stimulated differentiation of neural tissue, haemopoiesis and myotubes. We did not observe any kind of stimulative coooperative action of FGF and NGF in differentiation process. So it seems that NGF hinders the stimulating effect of FGF. NGF alone impaired growth of explants, but in combination with FGF acted synergistically, thus improving growth rate of cultivated embryos. Additional activation of gene with 5-azaC had no effect on possible NGF influence on neural tissue differentiation , but it resulted in improved myotubes differentiation. Activation of genes with FGF/5azaC signal as well as 5azaC/FGF/NGF combination improved the proportion of neural tissue and myotubes as well as haemopoieses. Obviously, these results confirm the role of FGF as neural inducer and mesoderm inducer. Anyway, FGF or NGF induced differentiation at least partially depend on status of gene methylation.

Kultura embrija sisavaca; fibroblastni faktor rasta; faktor rasta živaca; embrionalni teratomi

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