Vpliv različnih formulacij TiO2 nanodelcev na preživetje, diferenciacijo in nevrodegenerativne spremembe pri človeških nevralnih celicah in vitro

Koren, Tina (author), Pavlin, Mojca (mentor), Lojk, Jasna (co-mentor)

Namen magistrskega dela je bil določiti ali izbrani TiO2 nanodelci (ND) (ANATAZ, P25, FG) vplivajo na celice - preživetje, diferenciacijo, nastanek reaktivnih kisikovih zvrsti (ROS), proteine, ki so povezani z nevrodegenerativnimi spremembami, na modelu človeških nevralnih celic SH-SY5Y. Po vzpostavitvi in optimizaciji protokola diferenciacije celic SH-SY5Y, smo diferenciacijo potrdili z določitvijo sprememb v izražanju markerjev diferenciacije (MAP-2, Lamin B, GAP-43) s SDS-PAGE ter prenosom western, kjer smo opazili statistično značilno povečanje vseh diferenciacijskih markerjev v primerjavi z nediferenciranimi celicami. Z uporabo fluorescentnih barvil Hoechst 33342 in PI ter fluorescenčne mikroskopije nismo opazili statistično značilnega vpliva TiO2 ND koncentracij 2, 10 in 25 µg/mL na preživetje diferenciranih celic SH-SY5Y po njihovi 1-dnevni in 5-dnevni izpostavljenosti ND. Prav tako s pomočjo SDS-PAGE ter prenosa western nismo opazili statistično značilnega vpliva TiO2 ND koncentracije 10 in 25 µg/mL na potek diferenciacije celic SH-SY5Y. S pomočjo spektrofluorometrične meritve smo opazili statistično značilno povečanje ROS pri najvišji dodani koncentraciji ND 25 μg/mL, tako pri 5-dnevni izpostavljenosti P25 in ANATAZ ND ter 1-dnevni izpostavljenosti P25 in FG ND. Z uporabo SDS-PAGE ter prenosa western smo opazili, da prihaja do manjše razlike v količini amiloidnega prekurzorskega proteina (APP) ter beta-amiloida (Aβ) po 5-dnevni in 1-dnevni izpostavljenosti celic ND koncentracije 25 µg/mL, ne moremo pa identificirati nastalih fragmentov APP, zato tudi ne moremo določiti pomena teh sprememb. V sklopu magistrskega dela smo uspešno vzpostavili protokol diferenciacije celic SH-SY5Y, pokazali vpliv izbranih TiO2 ND na nastanek ROS ter na količino znotrajceličnih proteinov APP in Aβ. Nismo pa opazili vpliva izbranih ND na preživetje ter diferenciacijo celic SH-SY5Y.

• Language: Slovenian
• Keywords: TiO2, nanodelci, SH-SY5Y, nevrodegeneracija, ROS, diferenciacija, preživetje
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2020
• PID: 20.500.12556/RUL-121792
• COBISS.SI-ID: 44391939
• Publication date in RUL: 30.10.2020

Secondary language

• Language: English
• Title: The influence of different formulations of TiO2 nanoparticles on survival, differentiation and neurodegenerative changes on human neural cells in vitro

Abstract

The purpose of this master's thesis was to determine whether selected TiO2 nanoparticles (NP) (ANATASE, P25, FG) affect cells - survival, differentiation, formation of reactive oxygen species (ROS), proteins associated with neurodegenerative changes, in the model of human neural cells SH-SY5Y. After establishing and optimising the SH-SY5Y cell differentiation protocol, the differentiation was confirmed by determining changes in the expression of differentiation markers (MAP-2, Lamin B, GAP-43) by SDS-PAGE and western blot, where we observed a statistically significant increase in all differentiation markers compared with undifferentiated cells. Using fluorescent dyes Hoechst 33342 and PI and fluorescence microscopy, no statistically significant effect of TiO2 NP concentrations of 2, 10, 25 µg/mL on the survival of differentiated SH-SY5Y cells was observed after their 1-day and 5-days exposure. Also, no statistically significant effect of TiO2 NP concentrations of 10 and 25 µg/mL on the course of SH-SY5Y cell differentiation was observed by SDS-PAGE and western blot. By means of spectrofluorometric measurement, a statistically significant increase in ROS was observed at the highest added NP concentration of 25 μg/mL, both at 5-days exposure to P25 and ANATASE NP and at 1-day exposure to P25 and FG NP. Using SDS-PAGE and western blot, we observed a small difference in the amount of amyloid precursor protein (APP) and beta-amyloid (Aβ) after 5-days and 1-day of cell exposure to NP at 25 µg/mL concentration, but we could not identify the resulting APP fragments, so nor can we determine the significance of these changes. As part of the master's thesis, we successfully established the SH-SY5Y cell differentiation protocol, showed the influence of selected TiO2 NP on ROS formation and the amount of intracellular proteins APP and Aβ. We did not observe any effects of selected NP on cell survival and differentiation.

• Keywords: TiO2, nanoparticles, SH-SY5Y, nevrodegeneration, ROS, differentiation, survival