20.500.12556/RUL-155142 Optimizacija postopka za gojitev krvnih celic repatih dvoživk in vitro Optimization of in vitro cultivation of the blood cells of urodeles Gojenje krvnih celic je pomemben nedestruktiven pristop pridobivanja mitotičnega materiala (tj. metafaznih kromosomov), ki se ga uporabi za študije citogenetike in evolucije. Zaradi nezadovoljivega števila metafaznih kromosomov v predhodnih poskusih in vitro na krvi močerila (Proteus anguinus), smo metodo v tej raziskavi dodatno optimizirali in vpeljali tudi gojenje krvnih celic modelne repate dvoživke (Pleurodeles waltl). Primarno celično kulturo smo vzpostavili iz frakcije levkocitov in polne krvi. S testom proliferacije z BrdU smo ugotovili, da je ustrezna dolžina inkubacije z mitogenom PHA-M, ki je potreben za proliferacijo limfocitov, 4-5 dni pri močerilu in manj kot 4 dni pri španskem pupku ter v koncentraciji 6 % (vol/vol). Ustrezna koncentracija antimitotika kolcemida je bila 3 in 8 μg/ml, dodanega v celično kulturo za 24 in 48 ur. Kot najbolj obetavni vzorci za gojenje in vitro so se izkazali krvni vzorci zaporednih odvzemov krvi pri isti živali v kratkem časovnem intervalu (od 7 do 21 dni), saj spodbudimo proliferacijo krvnih celic in vivo. Na ta način smo pri španskem pupku uspeli pridobiti visoko število setov metafaznih kromosomov (več sto na eno objektno steklo) in zadovoljivo število setov metafaznih kromosomov pri močerilu (do 10). Vseeno pa je potrebna nadaljnja optimizacija postopkov po zaključku gojitve celic in vitro, da bo zagotovljena tudi ustrezna razporeditev in morfologija kromosomov za citogenetske analize. Culturing blood cells is an important non-destructive approach for obtaining mitotic material (i.e. metaphase chromosomes) that can be used for cytogenetic and evolutionary studies. As the number of metaphase chromosomes was to low in previous in vitro experiments with blood of the olm (Proteus anguinus), the method was further optimized. We also included another urodele, the Iberian ribbed newt (Pleurodeles waltl), as a model organism in the study. The primary cell culture was established from the leukocyte fraction and from whole blood. Based on the BrdU proliferation assay, we found that the incubation time required for lymphocyte proliferation with the mitogen PHA-M was 4–5 days in the olm and less than 4 days in the Iberian ribbed newt, and at a concentration of 6% (vol/vol). The appropriate concentration of the antimitotic colcemid was 3 and 8 µg/ml, which was added to the cell culture for both 24 and 48 hours. The most promising samples for in vitro cultures were blood samples after in vivo stimulation of cell proliferation with sequential blood sampling in a short time interval (from 7 to 21 days). With this type of blood samples, we were able to obtain a high number of metaphase chromosome sets in the Iberian ribbed newt (several hundred per slide) and a satisfactory number of metaphase chromosome sets in the olm (up to 10). However, further optimization of the procedures after in vitro culturing is required to ensure adequate distribution and morphology of chromosomes spread for cytogenetic analyzes. Proteus anguinus Pleurodeles waltl krvne celice citogenetika Proteus anguinus Pleurodeles waltl blood cells cytogenetics true false false [V. Forte Berlec] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2024-03-21 11:49:13 2024-03-21 11:49:19 2024-04-19 10:32:19 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 575:597.6/.9(043.2) 236374 193116931 39108.pdf 39108.pdf 1 01B34A90A1C6BBD04A26B39524AE8179 9f1cfa73367b14f3c03958b25536dcbcd2c0111efed538855cd1b60e3cec4e7c ac850c82-e770-11ee-922d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=181933 Biotehniška fakulteta 0 0 0