Lipid rafts isolation before and after myelination process in brain of wild type mouse C57Bl6

Uvod: Mijelin je izolacijska membrana koja je koncentrično omotana oko aksona. Ona sadrži tri vrste proteina: glikoprotein vezan za mijelin (MAG), bazični mijelinski protein (MBP) i proteolipidni protein (PLP) te lipide i kolesterol. Lipidi se nalaze u lokaliziranim regijama koje se nazivaju lipidne splavi. Glavne molekule koje izgrađuju lipidne splavi su kolesterol, sfingomijelin i glikosfingolipidi, posebice gangliozidi. Gangliozidi osiguravaju pravilnu funkciju lipidnih splavi te im se povećava koncentracija starenjem mozga. Marker splavi je flotilin, a nesplavi transferin. Ciljevi: Izolirati lipidne splavi iz mozgova miševa divljeg tipa (engl. wild type, WT) C57BI6 prije (5 dana starosti), tijekom (15 dana starosti) i nakon završenog procesa mijelinizacije (30 dana starosti) te utvrditi kako se pojavljuju markeri mijelina u lipidnim splavima. Materijali i metode: U istraživanju su korišteni uzorci moždanog tkiva divljeg tipa miša C57BI6 različite starosti (5 dana, 15 dana i 30 dana starosti). Nakon potvrde genotipa životinje su žrtvovane, a uzorci mozgova su smrznuti u tekućem dušiku. Iz smrznutih uzoraka izolirano je moždano deblo te je provedena izolacija lipidnih splavi ultracentrifugiranjem u gradijentu saharoze. Sastav dobivenih frakcija odredio se Western blot metodom korištenjem markera splavi i nesplavi te markera mijelina MAG i MBP. Rezultati su analizirani u računalnom programu Fiji i IBM SPSS Statistics v22. Rezultati: Istraživanjem je utvrđena statistički značajna razlika u izražaju proteina flotilina, transferina i MBP-a u moždanom deblu miševa starih 5, 15 i 30 dana. Izmjerene koncentracije kolesterola pokazuju niske vrijednosti. Zaključak: Sastav lipidnih splavi moždanog debla miša mijenja se tijekom razvoja i diferencijacije što dobro prikazuju markeri flotilin i transferin.Introduction: Myelin is an insulation membrane that is wrapped in concentric circles around the axons. It contains three types of protein: myelin-associated glycoprotein (MAG), myelin basic protein (MBP) and proteolipid protein (PLP), as well as lipids and cholesterol. Lipids are located in localized regions commonly known as lipid rafts. The main molecules that form lipid rafts are cholesterol, sphingomyelin, glycosphingolipids, and especially gangliosides, which ensure the proper function of lipid rafts and whose concentration is increased with brain aging. Flotillin and transferrin are the markers of lipid rafts and lipid non-rafts respectively. Research objectives: To isolate lipid rafts from brains of C57BI6 wild-type mice (WT) before (5 days of age), during (15 days of age) and after the completion of the myelination process (30 days of age) and to determine the manner of appearance of myelin markers in lipid rafts. Materials and methods: The research was carried out using samples of brain tissue of C57BI6 wild-type mice of different age (5, 15 and 30 days of age). After confirming the genotype, the animals were sacrificed and the mice brain samples were frozen in liquid nitrogen. The brain stem was isolated from the frozen samples and the lipid rafts were isolated by sucrose gradient centrifugation. The composition of the obtained fractions was determined by the Western blot method using markers of rafts and non-rafts as well as MAG and MBP myelin markers. The results were analyzed using the computer programs Fiji and IBM SPSS Statistics v22. Results: The research has shown a statistically significant difference in results of proteins flotillin, transferrin and MBP in brain stems of mice of 5, 15 and 30 days of age. The measured concentrations of cholesterol show low values. Conclusion: The composition of the lipid rafts of the mouse brainstem changes during development and differentiation, which are well demonstrated by the flotillin and transferrin markers