Analysis of Fibroblast Growth Factor Influence on Growth and Developmental Potential of Rat Foetuses in the In Vitro Culture Model

The fibroblast growth factor’s (FGF) influence on the growth and differentiation of 8- and 9- day-old rat foetus has been studied, whereas foetuses were grown in an in vitro culture model. Proliferation was analysed by the expression of proliferating cell nuclear antigen (PCNA). It was established that the usage of FGF in the first period of the culture lowers the growth no matter the foetus age at the moment of culturing and no matter whether it is a medium with or without a serum. If FGF is applied in the second culture period, it also lowers the growth, however younger foetuses in the in vitro culture model are more sensible to FGF negative influence.When FGF was applied in a lower concentration the growth of whole foetuses was improved in the in vitro culture model, which shows that the FGF influence on growth depends on the concentration. Stereological analyses have been done and showed that, in the in vitro culture model, FGF has no influence on proliferating cartilage tissue, but it stimulates the survival of nervel tissue cells. It has been shown that the quantitative research of growth processes in cultivated foetuses can precisely be done by combining classic methods of measuring whole foetus diameters and analysing the expression of proliferating antigen

Lymph Node, Spleen and Peripheral Blood Lymphocytes as Stimulators of Alloreactivity

Before and after kidney transplantations, in vitro tests that measure the level of reactivity between donor and recipient lymphocytes are performed for better organ selection and as indicator of possible organ rejection. In these tests, donor’s and recipient’s lymphocytes are stimulated for proliferation, which intensity is measured and accordingly organ recipient reactivity towards graft is determined. Lymph node, spleen and peripheral blood lymphocytes are used for those purposes. For better interpretation of these in vitro tests it should be important to determine mitogenic ability of lymphocytes of different origin and to choose the most adequate cells. To compare mitogenic ability of deceased donor lymph node, spleen and peripheral blood lymphocytes one-way mixed lymphocyte culture (MLC) was used. As stimulators irradiated lymphocytes from spleen, lymph node and peripheral blood samples of 12 deceased donors were used while as responders lymphocytes from peripheral blood of healthy individuals, chosen according HLA-DRB1 alleles (stimulators and responders were HLA-DRB1 identical, semi-identical or different), were used. Spleen lymphocyte activity was the best with different cells and the weakest with identical cells. Impact of polyclonal mitogens (PHA – phytohemagglutinin, Con A – concanavalin A and PWM – pokeweed mitogen) on lymphocyte proliferation was tested on lymphocytes from spleen and lymph node of deceased donors. Results abtained in culture in vitro showed that spleen cells had exerted the best mitogenic potential and PHA had the greatest impact upon lymphocyte proliferation. This investigation is of importance for establishing the best model to reflect in vivo situation in transplanted patient

Genomic Instability of the APC Gene Found in Glioblastoma

The etiology and pathogenesis of tumors of the central nervous system are still inadequately explained. This study analyses tumor suppressor gene— adenomatous polyposis coli (APC) in 28 patients with glioblastoma, the most aggressive form of glial tumors. APC protein has a structural role in adherens junctions, but also plays a signaling role as a negative regulator of the wnt pathway. Our interest in the APC gene stemmed principally from the findings that the wild-type APC protein is highly expressed in the central nervous system, and upon the finding that it is critically involved in particular syndromes, among which brain tumors play a significant role. Glioblastoma samples were tested for gene instability by PCR/loss of heterozygosity using RFLP method. Two polymorphic markers were used: an Rsa I polymorphic site in exon 11, and an Msp I polymorphic site in exon 15. The results of our analysis for both markers showed allelic loss of the APC gene in 40% of our sample out of 25 heterozygous patients (informativeness 89%). Another 20% of samples demonstrated allelic imbalance of the APC allele in tumor tissue. Altogether, there were 15 samples (60%) demonstrating instability of this tumor suppressor gene. Despite increasing knowledge on glioma biology and genetics, the prognostic tools for glioblastoma still need improvement. Our findings on genomic instability of the APC gene may contribute to better understanding of glioblastoma genetic profile and could be used as a prognostic marker of disease evolution and progression

Preživljenje fetusa određeno utjecajem 5-azacitidina na placentu

DNA methylation as a regulatory mechanism for mammalian gene expression is involved in the process of placentation as well as foetal development. 5azaC is a demethylating agent, which incorporates into DNA instead of cytosine and prevents its methylation, subsequently changes expression of genes involved in normal placental and foetal development. Single dose of 5azaC (5 mg/kg) was administered to pregnant rats at 11th, 12th and 13th day of gestation. Placentas and foetuses were isolated at day 20, weighted and histologicaly analyzed. After 5azaC administration on the 11th day of gestation, treated placentas were significantly smaller and labyrinth was significantly reduced. Consequently complete foetal resorption occurred. When administrated at day 12 of gestation, labyrinth was slightly recovered and 24% foetuses survived, while after its application at 13th day of gestation, almost normal distribution of placental layers was found and survival was 96%. These results confirmed epigenetic influence upon placental development. 5azaC caused changes in its structure, especially the reduction of labyrinth that is crucial for foetal survival. After establishment of normal placental layers distribution, 5azaC has no impact on placental morphology, neither on foetal survival. On the other hand, the influence of 5azaC remains visible in appearance of foetal malformations.Metilacija DNA kao regulatorni mehanizam ekspresije gena u sisavaca uključena je kako u procese placentacije tako i razvitka fetusa. Demetilacijsko sredstvo 5azaC ugrađuje se u DNA umjesto citozina i sprječava njegovu metilaciju, što ima za posljedicu promijenjenu ekspresiju gena uključenih u normalnu placentaciju i fetalni razvitak. Trudne ženke Fisher soja štakora tretirane su s 5 mg/kg 5azaC jedanaestog, dvanaestog i trinaestog dana gestacije. Dvadesetog dana gestacije izolirani su, izvagani i histološki analizirani kako fetusi tako i placente. Nakon primjene 5azaC jedanaestog dana gestacije, tretirane placente bile su značajno manje, a labirint reduciran, zbog čega je uslijedila potpuna resorpcija fetusa. Dvanaesti dan primjene 5azaC rezultirao je blagim povećanjem labirinta, a preživljenje fetusa bilo je 24%. Tek trinaestog dana aplikacije 5azaC uspostavila se normalna distribucija labirinta i bazalnog sloja s preživljenjem od 96%. Dobiveni rezultati potvrđuju epigenetski utjecaj na razvitak placente. Primjena 5azaC uzrokovala je promjene u strukturi placente, osobito redukciju labirinta čija je uloga očito ključna za preživljenje fetusa. Nakon uspostave normalne građe placente, 5azaC više nije imao utjecaja na njezinu morfologiju niti na preživljenje fetusa. S druge strane, njegov je teratogeni utjecaj ostao i dalje vidljiv na fetusima u obliku različitih malformacija

Preživljavanje embrijskog tkiva štakora nakon ektopične transplantacije

Investigation of the developmental potential of embryonic cells is important in designing novel approaches in the field of regenerative medicine. The purpose of our experiments was to compare the survival of different embryonic tissues transplanted at an ectopic site in vivo. Embryo proper (9.5-day-old), neural retinas (20-day-old), lensectomized eyes (14- and 18-day-old), epiglottis (17-day-old), mandible (13- and 14-day-old), lacrimal gland (17- and 20-day-old) were microsurgically isolated from rat embryos and transplanted under the renal capsule. The embryo-proper survived in transplants for at least 60 days. Fetal rat retina survived in transplants for 180 days forming rosettes. Neural and glia cells with abundant neuropil as well as plexiform layers were found by electron microscopy. The lensectomized eye survived in transplants for 66 days. Transdifferentiation of the retina to lens cells was discovered. The epiglottis, mandible and lacrimal gland survived in transplants for at least 14 days. In the mandible, fully structured teeth developed. This study showed not only the early postimplantation embryo but also various more developed tissues and organs to be able to survive under the renal capsule for at least 14 days. This ectopic site has therefore proved to be a very convenient environment for transplantation experiments.Istraživanje razvojnoga potencijala embrijskih stanica osnova je novih pristupa terapiji u području regenerativne medicine. U našim istraživanjima proučavali smo preživljenje različitih embrijskih tkiva nakon ektopične transplantacije in vivo. Zametak u užem smislu starosti 9,5 dana, neuralna mrežnica (20 dana), lensektomirano oko (14 i 18 dana), epiglotis (17 dana), mandibula (13 i 14 dana) te suzna žlijezda (17 i 20 dana) mikrokirurški su izolirane iz štakorskih zametaka navedenih starosti i transplantirane pod bubrežnu čahuru odraslih štakora. Zametak je u transplantatu preživio 60 dana. Fetalna mrežnica preživjela je čak 180 dana stvarajući rozete u transplantatu. Elektronskom mikroskopijom u njoj su dokazane živčane i glija stanice s obilnim neuropilom i mrežastim slojem. Lensektomirano oko preživjelo je 66 dana, a u transplantatu je otkrivena transdiferencijacija mrežnice u stanice leće. Epiglotis, mandibula i suzna žlijezda preživjele su 14 dana u transplantatu. U mandibuli su se razvili dobro formirani zubi. Pokazali smo, dakle, da uz rani poslijeimplantacijski zametak štakora, različita druga diferenciranija tkiva i organi zametka mogu također preživjeti pod bubrežnom čahurom najmanje 14 dana. Ovo ektopično mjesto ponovno se je dokazalo kao vrlo pogodno za pokuse transplantacije

Ekspresija nuklearnog antigena stanične proliferacije i proteina retinoblastoma u presađenoj fetalnoj suznoj žlijezdi štakora

A model of ectopic organogenesis of the rat fetal lacrimal gland was developed to study lacrimal gland organogenesis. It was done by its transplantation under the renal capsule. In transplants, expression of the tumor suppressor retinoblastoma gene (Rb) and proliferation cell nuclear antigen (PCNA) was assessed at the protein level. Eyes of 17- and 20-day-old rat fetuses were isolated under a dissecting microscope. Lacrimal glands were found and transplanted under the renal capsule of an adult male. After 14 days, transplants were routinely prepared for immunohistochemistry. DAKO Animal Research kit (Peroxidase) was used for detection of monoclonal mouse anti-PCNA and monoclonal mouse anti-human retinoblastoma gene product. In transplants, teratoma-like structures developed that contained lacrimal gland epithelial cells and ducts as well as epidermis. PCNA signal was detected in the nuclei of excretory duct cells and in epidermal basal layer cells. Some transplant cells were found to have the ability of proliferation preserved even after a 14-day period. PCNA signal was absent in well differentiated epithelial cells of lacrimal gland. Intranuclear Rb protein expression was only detected in several scattered cells, indicating the proliferating compartment to be usually larger than the differentiating one in fetal tissues. Assessment of the PCNA and Rb gene expression could prove important for elucidation of pathologic processes of the lacrimal gland, such as tumors or dry eye syndrome.Razvijen je model ektopične organogeneze fetalne suzne žlijezde štakora za ispitivanje organogeneze suzne žlijezde. To je postignuto presađivanjem suzne žlijezde ispod bubrežne kapsule. U presatcima je procjenjivana ekspresija gena tumorske supresije retinoblastoma (Rb) i jezgrenog antigena proliferirajućih stanica (PCNA) na razini bjelančevina. Oči fetusa štakora starih 17 i 20 dana izolirane su pod disekcijskim mikroskopom. Suzne žlijezde su pronađene i presađene pod bubrežnu kapsulu odraslog mužjaka. Nakon 14 dana su presatci rutinski pripravljeni za imunohistokemijsku analizu. Za otkrivanje monoklonskog mišjeg anti-PCNA i monoklonskog mišjeg anti-humanog proizvoda gena retinoblastoma uporabljen je DAKO Animal Research test (peroksidaza). U presatcima su se razvile teratomu slične strukture koje su sadržavale epitelne stanice suzne žlijezde i kanale, te epidermis. Signal PCNA otkriven je u jezgrama stanica odvodnog kanala, te u stanicama epidermnog bazalnog sloja. Time je pokazano kako su neke stanice presatka zadržale sposobnost proliferacije i nakon 14-dnevnog razdoblja. Signal PCNA bio je odsutan u dobro diferenciranim epitelnim stanicama suzne žlijezde. Unutarstanična ekspresija proteina Rb otkrivena je samo u nekoliko razasutih stanica, što pokazuje da je proliferirajući odjeljak u fetalnim tkivima obično veći od diferencirajućeg odjeljka. Procjena ekspresije PCNA i gena Rb mogla bi biti važna za pojašnjenje patoloških procesa u suznoj žlijezdi, kao što su tumori ili sindrom suhog oka