magistrsko delo
Tomaž Leskovar (Avtor), Lea Rems (Mentor), Anja Blažič (Komentor)

Povzetek

Z elektroporacijo povečamo prepustnost plazemske membrane, kar nam omogoči, da v celico vnesemo ali iz nje ekstrahiramo želene učinkovine, ki plazemske membrane sicer ne prehajajo. Ta tehnika se uporablja v različnih medicinskih aplikacijah, kot so gensko zdravljenje, zdravljenje tumorjev in dostava zdravil. Uspešnost elektroporacije temelji na doseganju ravnotežja med povišanjem prepustnosti plazemske membrane in preživetjem celic. Pomembno vlogo pri preživetju celic predstavlja sposobnost celice, da obnovi mirovno transmembransko napetost in vzpostavi celično homeostazo. V nalogi smo raziskovali, kako elektroporacija vpliva na spremembe v transmembranski napetosti pri ovarijskih celicah kitajskega hrčka (CHO-K1) in kako na te spremembe vplivata zaviralec TRPM4 kanalov 9-hydroxyphenanthrene (9-phenantrol) in zaviralec kalijevih ionskih kanalov tetraetilamonij (TEA). Predpostavili smo, da bi zaviralec 9-phenantrol lahko imel vpliv na spremembe v TMN, saj so predhodne študije pokazale, da CHO celice izražajo endogene TRPM4 kanale. Zaviralec TEA pa je bil izbran kot negativna kontrola, saj CHO celice ne izražajo znatnega števila kalijevih kanalov. Elektroporacijo smo dosegli z izpostavitvijo celic enemu električnemu pulzu dolžine 10 µs, 100 µs ali 1000 µs. Amplitudo pulza smo določili na podlagi meritev vnosa kalcija v celice s pomočjo fluorescenčnega barvila Fluo4-AM. Za vsako dolžino pulza smo določili najnižjo amplitudo, pri kateri smo zaznali znatni vnosa kalcija v celice. Poleg te najnižje amplitude, ki predstavlja prag za elektroporacijo, smo izbrali še približno 2-krat višjo amplitudo. Pulze izbranih amplitud smo nato uporabili pri nadaljnjih meritvah sprememb v transmembranski napetosti. Za slednje smo uporabili potenciometrično barvilo FLIPR, katerega fluorescenca se spreminja s spremembo v TMN. Ugotovili smo, da se membrana po elektroporaciji depolarizira, vendar nobeden od testiranih zaviralcev ni imel signifikantnega vpliva na to depolarizacijo, ne glede na dolžino in amplitudo pulza. To nakazuje, da je bila depolarizacija v veliki meri povzročena zaradi neselektivnega povečanja prepustnosti membrane zaradi elektroporacije. Za lažjo interpretacijo eksperimentalnih rezultatov smo razvili teoretični model, s katerim smo preverili, v kolikšni meri bi TRPM4 kanali sploh lahko prispevali k spremembam TMN po elektroporaciji. Model je potrdil, da TRPM4 kanali nimajo bistvenega vpliva na depolarizacijo, saj se pri danih pogojih membrana povsem depolarizira že zaradi neselektivnega toka ionov prek por v elektroporirani membrani. Poskusi so razkrili še, da nizkonapetostni pred-pulz dolžine 45 ms, ki ga proži elektroporator BTX Gemini za meritev upornosti vzorca pred proženjem elektroporacijskega pulza, zmanjša fluorescenco barvila FLIPR. Mehanizmi tega vpliva niso povezani s povečanjem prepustnosti membrane in ostajajo nepojasnjeni. V nadaljnjih poskusih bi bilo priporočljivo uporabljati elektroporator, ki pred dovajanjem pulza ne proži pred-pulza, tako da celice ne bi po nepotrebnem izpostavljali stresu, ki lahko vpliva na rezultat elektroporacije. Hkrati bi bilo smiselno testirati druga barvila za merjenje sprememb v transmembranski napetosti, ki bi lahko nadomestila barvilo FLIPR.

Ključne besede

elektroporacija;ovarijske celice kitajskega hrčka (CHO);transmembranska napetost;znotrajcelični kalcij;tetraetilamonij (TEA);9-hydroxyphenanthrene (9-phenantrol);magisteriji;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UL FE - Fakulteta za elektrotehniko
Založnik: [T. Leskovar]
UDK: 602.621(043.3)
COBISS: 180794883 Povezava se bo odprla v novem oknu
Št. ogledov: 104
Št. prenosov: 16
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Monitoring changes in transmembrane voltage after in vitro cell electroporation in the presence of ion channel inhibitors
Sekundarni povzetek: Electroporation increases the permeability of the plasma membrane, allowing us to introduce or extract desired substances into/from the cell that would not normally pass through the plasma membrane. This technique has various medical applications such as gene therapy, tumor treatment, and drug delivery. The success of electroporation is based on achieving a balance between increasing the permeability of the plasma membrane and cell survival. A crucial role in cell survival is played by the cell's ability to restore the resting transmembrane potential and establish cellular homeostasis. In the thesis, we investigated how electroporation affects changes in transmembrane voltage in Chinese hamster ovary (CHO-K1) cells and how these changes are influenced by the TRPM4 channel inhibitor 9-hydroxyphenanthrene (9-Phenanthrene) and the potassium ion channel inhibitor tetraethylammonium (TEA). We hypothesized that the inhibitor 9-Phenanthrene might have an effect on the changes in TMN, as previous studies have shown that CHO cells express endogenous TRPM4 channels. However, the TEA inhibitor was chosen as a negative control as CHO cells do not express a significant number of potassium channels. Electroporation was achieved by exposing the cells to a single electrical pulse of 10 µs, 100 µs or 1000 µs. The amplitude of the pulse was determined from measurements of calcium uptake into the cells using the fluorescent dye Fluo4-AM. For each pulse length, we determined the lowest amplitude at which significant calcium uptake into the cells was detected. In addition to this lowest amplitude, which represents the threshold for electroporation, we selected an amplitude approximately 2x higher. Pulses of the selected amplitudes were then used in further measurements of changes in transmembrane voltage. For the latter, we used the FLIPR potentiometric dye, whose fluorescence changes with the change in TMN. We found that the membrane depolarizes after electroporation, but none of the inhibitors tested had a significant effect on this depolarization, regardless of the pulse length and amplitude. This suggests that the depolarization was largely caused by a non-selective increase in membrane permeability due to electroporation. To facilitate the interpretation of the experimental results, we developed a theoretical model to test the extent to which TRPM4 channels could contribute at all to the changes in TMN after electroporation. The model confirmed that TRPM4 channels do not have a significant effect on depolarization, as under the given conditions the membrane is already fully depolarized due to non-selective ion flux through the pores in the electroporated membrane. Experiments further revealed that a low-voltage pre-pulse of 45 ms, delivered by a BTX Gemini electroporator to measure the sample resistance before delivering the electroporation pulse, reduces the fluorescence of the FLIPR dye. The mechanisms of this effect are unrelated to the increase in membrane permeability and remain unexplained. In further experiments, it would be advisable to use an electroporator that does not deliver a pre-pulse before the electroporation pulse, to avoid unnecessarily exposing the cell to stress that may affect the electroporation result. At the same time, it would be useful to test other dyes to measure changes in transmembrane voltage that could replace the FLIPR dye.
Sekundarne ključne besede: electroporation;Chinese hamster ovary (CHO) cells;transmembrane voltage;intracellular calcium;tetraethylammonium (TEA);9-hydroxyphenanthrene (9-Phenanthrene);
Vrsta dela (COBISS): Magistrsko delo/naloga
Študijski program: 1000316
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Fak. za elektrotehniko
Strani: XI, 75 str.
ID: 21548304