[doktorska disertacija
Izak Patrik Miller (Author), Irina Milisav (Mentor)

Abstract

Hepatociti so visoko specializirane polarizirane jetrne celice, v katerih se vrši večina za jetra specifičnih funkcij. Izolacija primarnih hepatocitov je skupek stresnih dejavnikov, med katerimi izstopa encimska razgradnja medceličnih stikov. Zaradi stresa ob izolaciji se v primarnih podganjih hepatocitih pojavi reverzibilna stresna prilagoditev, imenovana predapoptotski celični odziv na stres (PACOS), ki preko inaktivacije kaspaze 9 onemogoči proženje apoptoze po mitohondrijski poti ter nastopi takoj po izolaciji in izzveni do 6. dne v kulturi. Namen našega dela je bil proučiti PACOS ter njegov vpliv na delovanje hepatocitov in na odziv hepatocitov na dodatni stresni dejavnik (hipoksija in reoksigenacija). Zanimalo nas je tudi, ali so pri proučevanju PACOS sveže tkivne rezine jeter kontrola celičnim kulturam primarnih hepatocitov. V doktorskem delu smo pokazali, da primarni hepatociti v kulturi preživijo ter ohranijo nivo presnovne aktivnosti in sposobnost tvorbe sečnine. Potrdili smo stalno pojavnost PACOS v izoliranih primarnih hepatocitih. Raziskali smo proženje apoptoze v podganjih primarnih hepatocitih in dokazali, da v normalnih pogojih poteče po mitohondrijski poti. Sveže jetrne rezine so glede PACOS lahko kontrola primarnim hepatocitom, saj je v rezinah kaspazo 9 moč učinkovito aktivirati. Prvič smo pokazali, da sta posledici stresa pri izolaciji hepatocitov dva reverzibilna odziva na stres: indukcija endogenega antioksidativnega odziva in PACOS. Oba sproži povečano nastajanje ROS, ki tako omogoči preživetje in normalno delovanje celic. Poleg tega smo pokazali, da so na stres prilagojeni primarni hepatociti manj občutljivi na proženje apoptoze po hipoksiji/reoksigenaciji kot hepatociti pozne kulture, pri katerih sta PACOS in antioksidativni odziv izzvenela. Primarni hepatociti so relevanten in z nekaterimi omejitvami ustrezen model za temeljno proučevanje detoksikacije, metabolizma in potencialnih terapevtikov. Na podlagi rezultatov naše raziskave poudarjamo, da se na stres prilagojeni hepatociti na dodatne stresne dejavnike odzivajo drugače kot hepatociti in vitro ter in vivo, v katerih stresnih odzivov ni. Razumevanje mehanizmov ROS in odzivov na stres v primarnih hepatocitih je ključnega pomena za načrtovanje raziskav, za interpretacijo rezultatov, za prenos izsledkov na razmere in vivo in v končni fazi za razumevanje fiziologije in patofiziologije jeter ter oblikovanje posegov za preprečevanje in zdravljenje jetrne patologije.

Keywords

predapoptotski celični odziv na stres;kaspaze;inaktivacija;hipoksija;apoptoza;mitohondriji;endogeni antioksidativni odziv;reaktive kisikove vrste;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL MF - Faculty of Medicine
Publisher: I. P. Miller
UDC: 576.36:611.36(043.3)
COBISS: 146613251 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Preapoptotic cell stress response in primary hepatocytes
Secondary abstract: Hepatocytes are highly specialized polarized liver cells in which most liver-specific functions are performed. Isolation of primary hepatocytes is a set of stressors, among which the enzymatic degradation of intercellular junctions stands out. Due to stress upon isolation, a reversible stress adaptation called pre-apoptotic cell stress response (PACOS) occurs in primary rat hepatocytes and through inactivation of caspase-9 prevents apoptosis triggering through the intrinsic pathway. PACOS is present immediately after isolation and dissipates by day 6 in culture. The aim of our study was to examine/investigate PACOS, its impact on hepatocyte function, hepatocyte response to additional stressors (hypoxia and reoxygenation), and whether precision-cut liver slices can be used as a control to primary hepatocyte cell cultures in the PACOS study. In our work we demonstrated that primary hepatocytes survive in culture and maintain the level of metabolic activity and the ability of urea production. We confirmed the constant occurrence of PACOS in isolated primary hepatocytes. We investigated the induction of apoptosis in primary hepatocytes and demonstrated that it is normally activated through the intrinsic pathway. Precision-cut liver slices can serve as a control to primary hepatocytes with respect to PACOS, as caspase-9 can be efficiently activated in the slices. For the first time, we have shown that the consequences of stress in hepatocyte isolation are two reversible responses to stress: induction of an endogenous antioxidant response and PACOS. Both are triggered by increased ROS, which mediate cell survival and normal function. In addition, we showed that stress-adapted primary hepatocytes are less susceptible to triggering apoptosis after hypoxia/reoxygenation than hepatocytes in which PACOS and antioxidant response are no longer present. Primary hepatocytes are a relevant and, with some limitations, appropriate model to study detoxification, metabolism, and potential therapeutics. Based on the results of our study, we emphasize that stress-adapted hepatocytes respond differently to additional stressors than hepatocytes in vitro and in vivo, in which the stress responses are not induced. Understanding the mechanisms of ROS and stress responses in primary hepatocytes is crucial for planning research, interpreting the results, applying them in vivo and, ultimately, for understanding liver physiology, pathophysiology, and devising interventions for prevention and treatment of liver pathology.
Secondary keywords: Hepatociti;Disertacije;Izolacija;Primarni hepatociti;Stres;Predapoptotski odziv;
Type (COBISS): Dissertation
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Medicinska fak.
Pages: X, 113, 14 f.
ID: 17798985