Učinek pripravka iz človeške amnijske membrane na vnetje in oksidativni stres na modelu intersticijskega cistitisa in vitro

Boc, Eva (author), Erdani Kreft, Mateja (mentor), Kuret, Tadeja (comentor)

Intersticijski cistitis (IC) je kronično vnetje sečnega mehurja, za katerega vzrok ni znan, patofiziologija pa ostaja ne povsem pojasnjena. V raziskavi smo želeli oceniti možnost uporabe izvlečka humane amnijske membrane (hAM) kot terapevtskega sredstva pri zdravljenju IC, kar smo preverjali z uporabo celične linije SV-HUC-1. In vitro model IC smo vzpostavili z dodatkom encima glukoza oksidaza (GO) v hranilni medij, kar je sprožilo oksidativni stres in vnetje. Proučevali smo 4 skupine različno tertiranih celic SV-HUC-1: (1) celice, izpostavljene GO prvih 24 ur od skupno 48 ur gojenja (kontrolna skupina); (2) celice, izpostavljene GO vseh 48 ur; (3) celice, izpostavljene prvih 24 ur GO in naslednjih 24 ur GO in izvlečku hAM; (4) celice, izpostavljene prvih 24 ur GO in naslednjih 24 ur izvlečku hAM. Z metodo qPCR smo analizirali izražanje genov, z metodami ELISA, s prenosom proteinov western in imunooznačevanjem pa izražanje proteinov, povezanih z vnetjem, oksidativnim stresom, celičnimi stiki in diferenciacijo. Med štirimi eksperimentalnimi skupinami je primerjava skupine, tretirane z GO, s kontrolno skupino potrdila uspešno sprožitev oksidativnega stresa in vnetja. Za oceno učinka hAM smo skupino, tretirano z GO in hAM, primerjali s skupino, tretirano le z GO po 48 urah. Protivnetni učinek je potrdilo statistično značilno manjše izražanje genov IL6, IL8, CXCL1, CXCL10, NFKB1, NFKB2, CD274 ter proteinov IL-6, IL-8, CXCL1 in pJAK1; pri genih STAT1, IRF9, IFNGR1, IFNGR2 ter proteinih CXCL10 in NF-κB p50 je bil prisoten trend zmanjšanja. Antioksidativnega učinka hAM nismo uspeli zanesljivo potrditi; izražanje SOD2 in CAT, se je sicer statistično značilno povečalo, v izražanju GSS in GPX1 je bil nakazan trend zmanjšanja, pri izražanju NFE2L2, NQO1 in HMOX1 pa ni bilo opaznih razlik. Da hAM spodbuja izražanje genov povezanih z diferenciacijo urotelijskih celic ter genov povezanih s celičnimi stiki sta potrdila statistično značilno večje izražanje TJP1 in KRT13. Z imunooznačevanjem smo potrdili statistično značilno večje izražanje proteinov ZO1 in KRT13. Naši rezultati potrjujejo protivnetni in delno tudi antioksidativni učinek hAM ter njen spodbuden učinek na obnovo medceličnih stikov in diferenciacijo urotelija.

• Language: Slovenian
• Keywords: humana amnijska membrana, izvleček, intersticijski cistitis, vnetje, oksidativni stres
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [E. Boc]
• Year: 2026
• PID: 20.500.12556/RUL-186322
• UDC: 616.62-002(043.2)
• COBISS.SI-ID: 289430275
• Publication date in RUL: 31.08.2026

Secondary language

• Language: English
• Title: Effects of human amniotic membrane derivative on inflammation and oxidative stress in in vitro model of interstitial cystitis

Abstract

Interstitial cystitis (IC) is chronic, non-bacterial inflammation of the urinary bladder. Its pathophysiology is not fully understood, and no causal treatment is currently available. This study evaluated the potential use of human amniotic membrane (hAM) extract in IC treatment. An in vitro IC model was established using immortalized human urothelial cells (SV-HUC-1) by adding glucose oxidase (GO) to the culture medium to induce oxidative stress. Four groups were studied: (1) cells exposed to GO for first 24 of total 48 hours (control), (2) cells exposed to GO for 48 hours, (3) cells exposed to GO for 24 hours followed by GO and hAM extract for the next 24 hours, (4) cells exposed to GO for first 24 hours followed by hAM extract for the next 24 hours. The expression of genes and proteines related to inflammation, oxidative stress, cell junctions, and differentiation was analysed by qPCR, ELISA, Western blot, and immunolabeling, respectively. Compared with the control group, GO treatment significantly induced oxidative stress and inflammation. The anti-inflammatory effect of hAM was demonstrated by a significant decrease in IL6, IL8, CXCL1, CXCL10, NFKB1, NFKB2, and CD274 gene expression, and reduced IL-6, IL-8, CXCL1, and pJAK1 protein levels. Genes STAT1, IRF9, IFNGR1, IFNGR2 and proteins CXCL10 and NF-κB p50 showed a decreasing trend. The antioxidant effect was less conclusive. While SOD2 and CAT expression increased significantly, GSS and GPX1 showed a decreasing trend, and no differences were observed for NFE2L2, NQO1, and HMOX1. hAM’s beneficial role in urothelial cell differentiation and junction integrity was supported by significant upregulation of TJP1 and KRT13, confirmed at the protein level by higher ZO1 and KRT13 expression. Overall, hAM demonstrated anti-inflammatory, partially antioxidant effects and promoted restoration of cell junctions and differentiation in urothelial cells, suggesting potential therapeutic relevance for IC.

• Keywords: human amniotic membrane, extract, interstitial cystitis, inflammation, oxidative stress