Vpliv TNF-α na nastanek tunelskih nanocevk v rakavih in normalnih urotelijskih celicah

Boštjančič, Ava (author), Resnik, Nataša (mentor)

Tunelske nanocevke (TNT) so dolgi, tanki cevasti celični izrastki, ki med seboj povezujejo celice ter jim omogočajo medcelično komunikacijo s prenosom celičnih in neceličnih sestavin. TNT sodelujejo v fizioloških in patoloških procesih, zato je razumevanje lastnosti TNT in vplivov na njihov nastanek ključnega pomena za razvoj tarčnih zdravil. V diplomskem delu smo želeli ugotoviti vpliv vnetnega citokina TNF-α, tumor nekrotizirajočega faktorja α, na nastanek TNT pri normalnih in rakavih urotelijskih celicah. Celično linijo normalnih urotelijskih celic SV-HUC-1 in rakavih urotelijskih celic T24 smo tretirali z 1, 10, 20, 30 in 50 ng/mL TNF-α 24 ur. TNT smo označili s faloidinom, ki se veže na aktinske filamente, in s fluorescenčno mikroskopijo analizirali število TNT ter dolžino TNT. Celice SV-HUC-1 in T24 so imele po tretiranju z vsemi koncentracijami TNF-α večje število TNT kot kontrolne celice. Statistično značilno povečanje števila TNT smo ugotovili pri tretiranju celic SV-HUC-1 z 1 ng/mL TNF-α, pri čemer se je število TNT povečalo trikrat v primerjavi s kontrolo. Statistično značilno povečanje števila nastalih TNT je pri celicah T24 povzročilo tretiranje z 20 in 30 ng/mL TNF-α, pri čemer se je število TNT povečalo dvakrat v primerjavi s kontrolo. V dolžini TNT ni prišlo do sprememb pri nobeni od celičnih linij po tretiranju s TNF-α. Po tretiranju s TNF-α smo s prenosom western analizirali TNFAIP2, s tumor nekrotizirajočim faktorjem α induciran protein 2, ki sodeluje pri signalizacijski poti TNF-α/NF-κB. Zmanjšano izražanje TNFAIP2 po 24-urnem tretiranju s TNF-α smo zaznali pri tretiranju celic SV-HUC-1 z 1 in 20 ng/mL TNF-α in povečano izražanje TNFAIP2 pri tretiranju celic T24 z 10 in 50 ng/mL TNF-α. Z izvedbo luminiscenčnega testa viabilnosti celic smo za uporabljene koncentracije TNF-α dokazali, da TNF-α nima citotoksičnega učinka. Celicam SV-HUC-1 se je viabilnost statistično značilno povečala pri tretiranju z 10 ng/mL TNF-α, celicam T24 pa pri tretiranju z 1, 10, 20, 30 in 50 ng/mL TNF-α. Ugotovili smo, da je TNF-α induktor nastanka TNT pri normalnih in rakavih urotelijskih celicah. S tem smo postavili izhodišče za nadaljnje raziskave vloge TNF-α pri medcelični komunikaciji s TNT pri urotelijskih celicah.

• Language: Slovenian
• Keywords: medcelično sporazumevanje, TNF-α, TNFAIP2, tunelske nanocevke, urotelijske celice
• Work type: Bachelor thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2026
• PID: 20.500.12556/RUL-186426
• Publication date in RUL: 01.09.2026

Secondary language

• Language: English
• Title: The effect of TNF-α on the formation of tunneling nanotubes in cancer and normal urothelial cells

Abstract

Tunneling nanotubes (TNTs) are long, thin tubular cell protrusions that connect cells and enable them to communicate with each other by transferring cellular and non-cellular components. TNTs participate in physiological and pathological processes, so understanding the properties of TNTs and the effects on their formation is crucial for the development of drugs for targeted delivery. In this thesis, we aimed to determine the influence of the inflammatory cytokine TNF-α, tumor necrosis factor α, on the formation of TNTs in normal and cancer urothelial cells. The cell line of normal urothelial cells SV HUC-1 and cancer urothelial cells T24 were treated with 1, 10, 20, 30 and 50 ng/mL TNF-α for 24 hours. TNTs were labeled with phalloidin, which binds to actin filaments, and the number of TNTs and the length of TNTs were analyzed by fluorescence microscopy. In SV-HUC-1 and T24 cells, the number of TNTs was higher than in control cells after treatment with all concentrations of TNF-α. A statistically significant increase in TNT number was observed when SV-HUC-1 cells were treated with 1 ng/mL TNF-α, with the number of TNTs increasing threefold compared to the control. A statistically significant increase in the number of TNTs formed was induced in T24 cells by treatment with 20 and 30 ng/mL TNF-α, with the number of TNTs increasing twofold compared to the control. No change in length of TNTs occurred in any cell line after treatment with TNF-α. TNFAIP2, tumor necrosis factor α induced protein 2, participates in the TNF-α/NF-κB signaling pathway, and its expression after treatment with TNF-α was analyzed by western blotting. Decreased TNFAIP2 expression was demonstrated in SV-HUC-1 cells when treated with 1 and 20 ng/mL TNF-α, while increased TNFAIP2 expression was demonstrated in T24 cells when treated with 10 and 50 ng/mL TNF-α. By performing a luminescent cell viability assay, we demonstrated that TNF-α has no cytotoxic effect for the used TNF-α concentrations. SV-HUC-1 cell viability was significantly increased by treatment with 10 ng/mL TNF-α, and T24 by treatment with 1, 10, 20, 30 and 50 ng/mL TNF-α. We found that TNF-α is an inducer of TNT formation in normal and cancer urothelial cells. This laid the foundation for further research into the role of the TNF-α in TNT mediated cell communication in urothelial cells.

• Keywords: intercellular communication, TNF-α, TNFAIP2, tunneling nanotubes, urothelial cells