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Življenje na zemlji je prilagojeno rotacijam našega planeta in njegovim 24-urnim ciklom izmenjevanja dneva in noči. To pomeni, da 24-urni cikel, ki je potreben, da se zemlja zavrti okoli svoje osi, določa delovanje in vedenje vseh organizmov na planetu. Prilagajanje ritmu zemlje živemu svetu omogoča notranja biološka ura, ki jo imenujemo cirkadiana ura. Gre za biokemijski oscilator, ki deluje v približno 24 ur trajajočih ciklih in je vezan na solarni čas. Človeško telo je kompleksen sistem, v katerem imajo različni organi natančno določene vloge, zato njihove celice izražajo različne gene, ki odgovarjajo na potrebe organizma. Izražanje perifernih cirkadianih genov vodi glavna ura, ta pa ni edina, ki lahko vpliva na izražanje v različnih tkivih. Namen magistrske naloge je bil ugotoviti, kako prisotnost holesterola in lipoproteinov HDL in LDL vpliva na izražanje izbranih genov cirkadiane ure in na metabolizem v izbranih humanih celičnih linijah. Izražanje genov smo spremljali z metodo RT-qPCR, pa tudi s spremljanjem izražanja luminiscenčnega reporterja v stabilno transficiranih celicah z uporabo aparature LumiCycle. S testom AlamarBlue smo spremljali tudi, kako lipidi in holesterol vplivajo na metabolično aktivnost celic. Ugotovili smo, da HDL nima bistvenega vpliva niti na metabolizem, niti na izražanje genov cirkadiane ure. Na drugi strani sta LDL in holesterol povzročila večje spremembe. LDL sicer ni močno vplival na metabolizem, je pa zmanjšal izražanje vseh cirkadianih genov, ki smo jih izmerili. Enako smo pokazali tudi pri holesterolu, ki je hkrati tudi pospešil metabolizem celic. Ugotovili smo tudi, da se vplivi obremenitev z lipidi razlikujejo pri različnih celičnih linijah, kar nakazuje, da je cirkadiano izražanje genov tkivno specifično. Naši rezultati kažejo, da je lipidni metabolizem tesno povezan s cirkadiano uro. To pomeni, da sodobni življenjski slog, ki vpliva tako na lipidni metabolizem, kot tudi na cirkadiano uro, lahko predstavlja velik zdravstveni problem. Life on Earth is adapted to the rotation of our planet and its 24 h light-dark cycles. This means that the 24 h cycle which is needed for one spin of the Earth determines functioning and behaviour of all organisms. Adapting to the Earth's rhythm is enabled by an internal biological clock, also known as the circadian clock. It is a biochemical oscillator, which acts in approximately 24 h cycles and depends on solar time. The human body is a complex system made of different organs with specific tasks, which means that they are expressing different genes, answering to specific body needs. Expressing of peripheral circadian genes is dictated by the core clock, but there are also other factors that can influence the expression of genes in different tissues as well. This masters degree research aimed to find out how cholesterol and lipoproteins HDL and LDL affect the expression of a set of circadian genes and metabolism in two selected human cell lines. Gene expression was measured with the RT-qPCR method and also by measuring the expression of luminescent reporter in stably transfected cells with the LumiCycle method. With the AlamarBlue test, we also measured how lipids and cholesterol affect the metabolic activity of the cells. We showed that HDL only marginally affects metabolism or circadian gene expression. On the other hand, LDL and cholesterol do have a major impact. LDL did not influence metabolism much but did cause a strong reduction of circadian gene expression of all selected circadian genes. We showed the same for cholesterol, which additionally strongly accelerated cell metabolism. We also showed that the effects of lipid load differ in different cell lines, which shows that circadian gene expression is tissue-specific. Our results show a tight connection between lipid metabolism and the circadian clock. This means that modern lifestyle which affects lipid metabolism and the circadian clock could become a major health issue. |
