Utjecaj promjene temperature i epigenetskih lijekova na razvoj pupoljaka udova štakora ex vivo [Effects of temperature changes and epigenetic drugs on ex vivo rat limb bud development]

Mužić, Vedrana (2015) Utjecaj promjene temperature i epigenetskih lijekova na razvoj pupoljaka udova štakora ex vivo [Effects of temperature changes and epigenetic drugs on ex vivo rat limb bud development]. PhD thesis, Sveučilište u Zagrebu.

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Abstract

The aim of the thesis was to create an ex vivo model of the mammalian limb bud development for research of temperature changes and epigenetic drugs without the influence of the maternal organism. 13 and/or 14 days old limb buds were microsurgically isolated from Fisher rat embryos and cultured from 3 to 14 days in culture with serum (Eagle's MEM with 50% rat serum) or chemically defined media (MEM, or MEM with defined supplements) at normothermia (37 ºC), the two-week hypothermia (31 ºC) and hyperthermia (43 ºC, 15 min applied at the beginning of culture). The overall growth of ellipsoidal explants was measured with an ocular micrometer, and the calculated area (A) was standardized on the day of plating (A0). The numerical density (Nv) of the mmunohistochemically detected marker of cell proliferation (PCNA) was assessed. Part of explants was transplanted under the kidney capsule of anaesthetized males. A classical light microscopy and statistical analysis of the results was performed. Ex vivo experiments revealed the growth and progress of differentiation into cartilage, epidermis and myotubes, while grafts of explants previously cultured in serum at normothermia expressed the residual potential for ossification and differentiation of skin appendages and nerves. Overall growth, Nv PCNA and histomorphology in the two-week normothermia conditions were the best in controls with serum and most affected in pure MEM. On hyperthermia, significant negative impact on the overall growth in the three-day cultures with serum was discovered, but Nv PCNA was significantly higher in both the serum supplemented and MEM culture conditions. In the two-week hypothermia overall growth did not differ depending on the media, or of normothermia. However, Nv PCNA was significantly lower than in the control with serum at normothermia. Epigenetic drug 5-azacytidine in comparison to controls reduced the growth and differentiation at all temperatures except at two-week normothermia in MEM (5μM), where the growth of the front limb buds was even significantly higher. NvPCNA was significantly lower in MEM with serum under the influence of 5-azaC. In prolonged hypothermia in MEM, 5-azaC reduces Nv PCNA while at hyperthermia it caused Summary necrosis in the three-day culture with serum (30μM). Trichostatin A (33nM) in MEM increased overall growth at normothermia, but without the possibility of survival for 14 days and also decreased growth, Nv PCNA and differentiation (no myotubes) at hyperthermia. TSA reduced Nv PCNA in a three-day culture at hyperthermia. In the original model that was established specific influences of temperature changes and epigenetic drugs on the parameters of the limb development, especially on survival, overall growth, and proliferative potential were discovered. That is of importance for a better understanding of possible side-effects of combined physical and pharmacological therapy.

Abstract in Croatian

Cilj disertacije bio je kreiranje ex vivo modela razvoja pupoljaka udova sisavca bez utjecaja organizma majke u svrhu istraživanja promjene temperature i epigenetskih lijekova. Pupoljci udova 13 i/ili 14 dana stari izolirani su mikrokirurški iz zametaka štakora Fisher i kultivirani od 3 do 14 dana u kulturi organa sa serumom (Eaglov MEM sa 50% štakorskog seruma) ili kemijski definiranim medijima (MEM ili MEM sa definiranim dodacima) na normotermiji (37 ºC), dvotjednoj hipotermiji (31 ºC) i hipertermiji (43 ºC, 15 min, početak kulture). Mjerio se ukupni rast elipsoidnih eksplantata okularnim mikrometrom, a izračunata površina (A) je normirana na dan nasađivanja (A0). Izračunata je numerička gustoća (Nv) imunohistokemijski detektiranog biljega stanične proliferacije (PCNA). Dio eksplantata transplantiran je pod čahuru bubrega anesteziranih mužjaka. Provedena je klasična svjetlosna mikroskopija i statistička obrada rezultata. U pokusima ex vivo otkriven je rast i napredak diferencijacije u hrskavicu, epidermis i miotube, a u transplantatima kultiviranim u serumu na normotermiji iskazao se ostatni potencijal za osifikaciju te diferencijaciju kožnih privjesaka i živaca. Ukupni rast, Nv PCNA i histomorfologija bili su na dvotjednoj normotermiji najbolji u kontrolama sa serumom, a najslabiji u čistom MEM-u. Na hipertermiji se iskazao negativni utjecaj na ukupni rast i to u trodnevnim kulturama sa serumom, ali je Nv PCNA bio značajno veći i u serumu i MEM-u. U dvotjednoj hipotermiji ukupan rast se nije razlikovao niti ovisno o mediju, a nije bio različit niti od onog na normotermiji. Ipak, Nv PCNA je bio značajno veći u serumu na kontrolnoj normotermiji. Epigenetski lijek 5-azacitidin u odnosu na kontrole smanjivao je rast i diferencijaciju na svim temperaturama osim na dvotjednoj normotermiji u MEM-u (5μM), gdje je rast prednjih pupoljaka čak značajno veći. Nv PCNA je značajno manji u MEM-u sa serumom pod utjecajem 5-azaC. U dugotrajnoj hipotermiji u MEM-u 5-azaC smanjuje Nv PCNA. U hipertermiji je uzrokovao nekrozu u trodnevnoj kulturi sa serumom (30μM). Trihostatin A (33nM) u MEM-u je povećao ukupni rast na normotermiji, ali bez mogućnosti preživljenja tijekom 14 dana, a smanjio rast, Nv PCNA i diferencijaciju na hipertermiji (nema miotuba). TSA je smanjio Nv PCNA na hipertermiji u trodnevnoj kulturi. U razvijenom originalnom modelu dokazan je specifičan utjecaj temperaturnih promjena i epigenetskih lijekova na parametre razvoja udova, posebno na preživljenje, ukupan rast i proliferacijski potencijal što je od značaja za bolje razumijevanje mogućih nuspojava udruženih fizikalnih i medikamentoznih terapija.

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