Vpliv temperature gojenja podganjih astrocitov v primarni kulturi na izražanje adrenergičnih receptorjev in monokarboksilatnih prenašalcev

Perko, Nika (author), Haque Chowdhury, Helena (mentor)

Astrociti so najštevilčnejše celice glija v možganih. Odgovorni so za obrambo in homeostazo centralnega živčnega sistema. Med povišano aktivnostjo nevronov, telovadbo ali nevropatološkim stanjem, je za njih značilna visoka stopnja glikolize z nastajanjem L-laktata, kar je znano kot Warburgov efekt. Pojav naj bi uravnavali noradrenegični nevroni, ki sproščajo noradrenalin, vpleteni pa so tudi adrenergični receptorji in monokarboskilatni prenašalci na membrani astrocitov. Vsa ta in številna druga dognanja so plod raziskav, ki so bile izvedene na celičnih kulturah. Protokoli za gojenje celičnih kultur se med laboratoriji lahko razlikujejo po sestavi gojišč in medijev ter pogoji okolja med gojenjem celic. Običajno se poskusi na astrocitih izvajajo na celičnih kulturah, gojenih pod pogoji, ki oponašajo stabilno naravno celično okolje (37 °C, 95 % vlažnost, 5 % CO2). Velikokrat pa se poskusi za nekaj ur izvajajo pri sobni temperaturi (22–24 °C). Zanimalo nas je, ali izpostavljenost astrocitov nižji temperaturi (34 °C) vpliva na izražanje adrenergičnih receptorjev 1A-AR in 2-AR ter monokarboksilatnh prenašalcev MCT1 in MCT4. Problema smo se lotili z imunocitokemično metodo in konfokalno mikroskopijo. Ugotovili smo, da je bilo izražanje 1A-AR manjše pri 34 °C kot pri 37 °C. Izražanje 2-AR pa je bilo pri nižji temperaturi večje. Pri izražanju genov za MCT1 in MCT4 smo opazili trend zmanjšane ekspresije pri nižji temperaturi, a bi za nedvoumno potrditev rezultatov morali izvesti dodatne poskuse. Čeprav smo poskuse izvajali na temperaturi višji od sobne in več časa, lahko sklepamo, da bi tudi nekajurna izpostavljenost sobnim temperaturam lahko imela podoben učinek. Primerno bi bilo izvesti dodatne poskuse pri nižji temperaturi in krajšem času izpostavljenosti ter preveriti še vplive drugih pogojev celičnega mikrookolja na izražanje genov preiskovanih proteinov.

• Language: Slovenian
• Keywords: astrociti, temperatura gojenja, izražanje proteinov, imunocitokemija, konfokalna mikroskopija
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-139938
• COBISS.SI-ID: 128610051
• Publication date in RUL: 08.09.2022

Secondary language

• Language: English
• Title: The effect of the culture temperature of rat astrocytes in primary culture on the expression of adrenergic receptors and monocarboxylate transporters

Abstract

Astrocytes are the most abundant glial cells in the brain. They are responsible for the defence and homeostasis of the central nervous system. During increased neuronal activity, exercise and pathophysiological conditions astrocytes are characterized by a high glycolytic rate with the formation of L-lactate, which is known as the Warburg effect. The phenomenon is thought to be regulated by noradrenergic neurons that release noradrenaline, and adrenergic receptors and monocarboxylate transporters on the membrane of astrocytes are also involved. All these and many other findings are the result of research conducted on cell cultures. Protocols for growing cell cultures may differ between laboratories with regard to the composition of culture media and media, and the environmental conditions during cell cultivation. Typically, experiments with astrocytes are performed in cell cultures grown under conditions that mimic a stable natural cell environment (37 °C, 95 % humidity, 5 % CO2). Experiments are often performed for several hours at room temperature (22–24 °C). We were interested in whether exposure of astrocytes to a lower temperature (34 °C) affects the expression of adrenergic receptors 1A-AR and 2-AR and monocarboxylate transporters MCT1 and MCT4. We tackled the problem employing immunocytochemical method and confocal microscopy. We found that the expression of 1A-AR was lower at 34 °C than at 37 °C. On the contrary, the expression of 2-AR was at 34 °C higher than at 37 °C. In the expression of the genes for MCT1 and MCT4, we observed a trend of decreased expression at lower temperature, but additional experiments should be performed to confirm the results unequivocally. Although we performed the experiments at a temperature higher than room temperature and for a longer time, we can conclude that even a few hours of exposure to room temperatures could have a similar effect. It would be appropriate to carry out additional experiments at a lower temperature and shorter exposure time, and to check the effects of other conditions of the cellular microenvironment on the gene expression of the investigated proteins.

• Keywords: astrocytes, culture temperature, gene expression, immunocytochemistry, confocal microscopy