Sestre Milosrdnice University hospital and Institute of Clinical Medical Research
Abstract
The aim was to analyze the regulation of growth and tissue differentiation in a unique in vitro4culture 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 the purpose of this study was to test their possible synergistic influence in the period when mammalian embryos are extremely sensitive to external factors. We cultivated 9.5-day-old rat embryos on metal grid supported lens paper, at the air-liquid interface in culture medium (Eagle\u27s minimal essential medium (MEM) with 50% of homologous serum) with the addition of FGF, NGF and a combination of FGF/NGF in a time frame of 9 days. Other three groups of embryos were for 24 hours pretreated with 5-azacytidine (5azaC), an agent that can activate repressed genes. A parallel group of nontreated control embryos were cultivated with each experimental group. During 14 days of culture embryos grown in teratoma-like explants and growth rate were evaluated by measuring average size of explants using an eyepiece micrometer on days 5, 7, 11 and 14 after the addition of growth factors. Differences between respective groups were estimated by Student\u27s t-test. Differentiated tissues were estimated on serial histologic sections. c2-test or Fisher exact test were used to compare the proportion of tissues between respective groups. In embryo-derived teratomas NGF or FGF/ NGF combination used within the 9 day time frame did not stimulate differentiation of any kind of tissues; moreover, FGF/NGF inhibited maturation of epidermis, while FGF stimulated differentiation of neural tissue, hemopoiesis and myotubes. We did not observe any kind of stimulative cooperative action of FGF and NGF in differentiation processes. 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 the growth rate of cultivated embryos. Additional activation of genes with 5azaC had no the effect on possible NGF influence on neural tissue differentiation, but resulted in improved myotube differentiation. The activation of genes with 5azaC/FGF signal and 5azaC/FGF/NGF combination improved the proportion of neural tissue and myotubes as well as hemopoiesis. Obviously, these results supported the role of FGF as neural inducer and mesoderm inducer. Anyway, FGF or NGF induced differentiation at least partially depends on the status of gene methylation.Namjera je bila istražiti regulaciju rasta i diferencijacije pomoću fibroblastnog faktora rasta (FGF) i faktora rasta živaca (NGF) u jedinstvenom in vitro modelu za kultiviranje embrija sisavaca u kritičnoj fazi razvoja, gastrulaciji, tijekom dva tjedna. Oba faktora imaju ključnu ulogu u embriogenezi sisavaca, a svrha je bila istražiti njihov mogući sinergistični učinak kada su embriji sisavaca najosjetljiviji na djelovanje pojedinih vanjskih čimbenika. Embrije štakora stare 9,5 dana kultivirali smo na metalnoj mrežici s lećnim papirićem, na granici plinske i tekuće faze, u temeljnom mediju za kultiviranje (Eagleovom minimalnom esencijalnom mediju (MEM) s 50% homolognog seruma) uz dodatak faktora rasta FGF, NGF i kombinacije FGF/NGF tijekom 9 dana kulture. Druge tri skupine embrija su prethodno bile tretirane 24 sata 5-azacitidinom (5azaC), agensom koji može aktivirati gene. Paralelne skupine embrija kultivirane su u temeljnom mediju kao kontrolne. Tijekom 14 dana kultiviranja embriji izrastu u teratoidne strukture, a pritom se rast procjenjuje određivanjem prosječene veličine eksplantata pomoću okularnog mikrometra u određene dane (5, 7, 11, 14) nakon dodavanja faktora rasta. Razlike u rastu između pojedinih skupina ispitane su Studentovim t-testom. Prisutnost diferenciranih tkiva u teratomima utvrđena je analizom na serijskim histološkim rezovima. Razlike u proporcijama diferenciranih tkiva između pojedinih skupina utvrđene su c2-testom ili Fisherovim egzaktnim testom. U embrijskim teratomima razvijenim in vitro niti NGF niti FGF/NGF kombinirani tretman nisu stimulirali diferencijaciju bilo koje vrste tkiva, dapače, FGF/NGF kombinacija je inhibirala sazrijevanje epidermisa. FGF je stimulirao diferencijaciju živčanog tkiva (p<0,05), miotuba i hemopoezu. Nismo mogli utvrditi sinergistični učinak FGF i NGF u procesu diferencijacije bilo koje vrste tkiva. Dapače, čini se da NGF smanjuje stimulacijski učinak FGF u procesu diferencijacije. NGF suzbija rast eksplantata, ali u kombinaciji s FGF djeluje sinergistično, značajno povećavajući rast eksplantata. NGF nije imao učinka na očekivano povećanje diferencijacije živčanog tkiva, nakon dodatne aktivacije gena pomoću 5azaC, međutim, diferencijacija miotuba je značajno povećana. Kombinirano djelovanje 5azaC/FGF i 5azaC/FGF/NGF na aktivnost gena povećalo je učestalost diferencijacije živčanog tkiva, miotuba i hemopoezu u eksplantatima. Ovaj rezultat potvrđuje djelovanje FGF kao induktora živčanog tkiva i mezoderma. U svakom slučaju diferencijacija potaknuta FGF i NGF signalima djelomice ovisi o stupnju metilacije gena
