Imunski odziv v steni sečnega mehurja miši po poškodbi s ciklofosfamidom

Hudobivnik, Laura (author), Zupančič, Daša (mentor)

Ciklofosfamid (CP) je alkilirajoči citostatik, ki se uporablja pri zdravljenju različnih vrst raka in avtoimunih obolenj. Njegov najpomembnejši neželeni učinek je hemoragični cistitis, ki nastane zaradi toksičnega metabolita akroleina. Kljub številnim raziskavam akutnega nebakterijskega cistitisa vloga limfocitov T pri imunskem odzivu v steni sečnega mehurja po poškodbi s CP še ni povsem pojasnjena. V diplomskem delu smo raziskali vpliv intraperitonealne (i.p.) aplikacije ciklofosfamida na prisotnost in porazdelitev limfocitov CD4+ in CD8+ v steni sečnega mehurja miši. Uporabili smo mišji model akutnega nebakterijskega cistitisa, pri katerem smo mišim injicirali enkratni odmerek 150 mg CP/kg telesne teže miši (skupina CP150) ali 300 mg CP/kg telesne teže (skupina CP300), kontrolna skupina pa je prejela i.p. injekcijo fiziološke raztopine. Analizirali smo histološke spremembe v steni mehurja na parafinskih rezinah, obarvanih s hematoksilinom in eozinom, prisotnost in lokalizacijo limfocitov T pa določili z imunofluorescenčnim označevanjem. Gostoto limfocitov T smo določili s kvantitativno analizo in ovrednotili s statistično analizo. Po aplikaciji CP smo opazili poškodbe urotelija in lamine proprije, med drugim luščenje urotelija, hiperplazijo, edem, vnetni infiltrat in hemoragijo. Morfološke spremembe so bile bolj izrazite pri skupini CP300. Z imunofluorescenčno mikroskopijo smo pokazali, da je bila v vseh skupinah lokalizacija limfocitov CD4+ in CD8+ pretežno v lamini propriji. Za nižji odmerek CP je bilo značilno povečanje števila limfocitov CD4+, medtem ko rezultati števila limfocitov CD8+ niso bili enoznačni. Pri višjem odmerku je prišlo do zmanjšanega števila obeh populacij limfocitov T. Statistična analiza je za limfocite CD4+ in CD8+ pokazala statistično značilne razlike med skupinama, ki sta prejeli različna odmerka CP, kar kaže na od odmerka odvisen učinek CP na lokalni imunski odziv. Za boljše razumevanje vloge limfocitov CD4+ in CD8+ pri cistitisu, induciranem s CP, so potrebne nadaljnje raziskave z večjim številom vzorcev, dodatnimi označevalci imunskih celic in molekularnimi metodami.

• Language: Slovenian
• Keywords: sečni mehur, mišji model akutnega nebakterijskega cistitisa, ciklofosfamid, imunski odziv, limfociti T, imunofluorescenca, mikroskopija
• Work type: Bachelor thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2026
• PID: 20.500.12556/RUL-186634
• Publication date in RUL: 03.09.2026

Secondary language

• Language: English
• Title: Immune response in the mouse urinary bladder wall following cyclophosphamide-induced injury

Abstract

Cyclophosphamide (CP) is an alkylating cytostatic agent used in the treatment of various cancers and autoimmune diseases. Its most significant adverse effect is hemorrhagic cystitis, caused by the toxic metabolite acrolein. Despite extensive research on acute non-bacterial cystitis, the role of T lymphocytes in the immune response of the urinary bladder following CP-induced injury is not yet fully understood. In this thesis, we investigated the effect of intraperitoneal (i.p.) injection of CP on the presence and distribution of CD4+ and CD8+ lymphocytes in the urinary bladder wall of mice. We employed a mouse model of acute non-bacterial cystitis, in which mice received a single i.p. injection of 150 mg CP/kg body weight (CP150 group) or 300 mg CP/kg body weight (CP300 group). The control group received an i.p. injection of physiological saline. Histological changes were evaluated on hematoxylin and eosin-stained paraffin sections, whereas the presence and localization of T lymphocytes were determined using immunofluorescence staining. T-lymphocyte density was quantified and evaluated using statistical analysis. Following CP administration, damage to the urothelium and lamina propria was observed, including urothelial desquamation, hyperplasia, edema, inflammatory infiltration, and hemorrhage. Morphological changes were more pronounced in the CP300 group. Immunofluorescence analysis demonstrated that across all groups, CD4+ and CD8+ lymphocytes were localized predominantly within the lamina propria. The lower CP dose was characterized by an increase in CD4+ lymphocytes, whereas the results regarding CD8+ lymphocytes were inconclusive. In contrast, the higher dose was associated with a reduction in both T lymphocyte populations. Statistical analysis identified significant differences for both CD4+ and CD8+ lymphocytes between groups treated with different CP doses, confirming a dose-dependent effect of CP on the local immune response. Further large-scale studies are needed to improve our understanding of the role of CD4+ and CD8+ lymphocytes in cyclophosphamide-induced cystitis.

• Keywords: urinary bladder, mouse model of acute non-bacterial cystitis, cyclophosphamide, immune response, T lymphocytes, immunofluorescence, microscopy