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Izražanje gena za (pro)katepsin X v celični liniji PC-12 ob prisotnosti amiloida beta
Šketelj, Blaž (Author), Doljak, Bojan (Mentor) More about this mentor... This link opens in a new window

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Abstract
V okviru magistrske naloge smo želeli preveriti ali citotoksični fragment Aβ(25-35) vpliva na izražanje gena za (pro)katepsin X v celični liniji PC-12. Celična linija PC-12 predstavlja dober model za preučevanje nevrodegeneracije, saj izraža tudi katepsin X v neaktivni obliki prokatepsina X, ki se nahaja v lizosomih. Nediferencirane celice PC-12 smo inkubirali z Aβ(25-35) v osmih časovnih intervalih (po 3 ure) in v lizatih celic s testom ELISA izmerili količino (pro)katepsina X ter s kinetično metodo encimske aktivnosti s fluorogenim substratom izmerili množino aktivnega katepsina X. Rezultate smo normirali na količino celokupnih proteinov, ki smo jih kvantificirali z metodo po Lowry-ju. Ugotovili smo, da se količina celokupnega katepsina X (pro in aktivna oblika) statistično značilno poveča že v 3 urah inkubacije z Aβ(25-35) in ostane povišana vse do 12 ur, kar bi lahko bilo posledica nespecifičnega stresnega odgovora celic na vstop amiloidnega peptida v celico. Izpostavitev Aβ(25-35) ne vpliva statistično značilno na množino aktivnega katepsina X v celicah PC-12, saj ostaja kljub povečanem izražanju gena za (pro)katepsina X, množina aktivnega katepsina X znotraj celic bolj ali manj konstantna. Morebitno izločanje aktivnega katepsin X iz celic v gojišče bi lahko potrdili z meritvijo encimske aktivnosti katepsina X v samem gojišču. Modelne celice PC-12 se torej odzivajo na prisotnost Aβ(25-35) v gojišču z nadizražanjem znotrajcelično izmerjenega prokatepsina X, kar bi lahko pomenilo nov molekulski mehanizem v obrambi pred amiloidom.

Language:Slovenian
Keywords:(pro)katepsin X, amiloid beta, celična linija PC-12, nevrodegeneracija
Work type:Master's thesis/paper (mb22)
Organization:FFA - Faculty of Pharmacy
Year:2020
Views:45
Downloads:45
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Secondary language

Language:English
Title:Expression of (pro)cathepsin X gene in the PC-12 cell line in the presence of amyloid beta
Abstract:
As part of the Master's thesis, we wanted to test whether cytotoxic fragment A affects the expression of the (pro) cathepsin X gene in PC-12 cell line. The PC-12 cell line represents a good model for studying neurodegeneration, as it also expresses the inactive form of the enzyme - procathepsin X in lysosomes. Undifferentiated PC-12 cells were incubated with A at eight time intervals (each 3 hours apart), the quantity of (pro) cathepsin X was determined by an ELISA assay and the amount of active cathepsin X was measured by kinetic method of enzymatic activity in cell lysates. The results were normalized to the total amount of proteins, quantified by the Lowry method. We found that the amount of total cathepsin X (pro and active form) increases statistically significantly within 3 h of incubation with Aβ(25-35) and remains elevated until 12 h, which could be due to the non-specific stress response of cells to the entry of the amyloid peptide into the cell. Exposure to Aβ(25-35) does not have a statistically significant effect on the amount of active cathepsin X in PC-12 cells. In spite of increased expression of the (pro) cathepsin X gene, the amount of active cathepsin X within the cells remains more or less constant. The possible excretion of active cathepsin X from cells into the culture medium could be confirmed by measuring the enzymatic activity of cathepsin X in the culture medium itself. The PC-12 model cells therefore respond to the presence of Aβ (25-35) in the culture medium by overexpressing intracellularly measured procathepsin X, which could represent a novel molecular mechanism in the defense against amyloid.

Keywords:(pro)cathepsin X, amyloid beta, PC-12 cell line, neurodegeneration

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