Ispitivanje protektivnog dejstva grafenskih kvantnih tačaka u modelu neuroinflamacije kod pacova
Examination of protective effect of graphene quantum dots in a model of neuroinflammation in rats
Author
Tasić, JelenaMentor
Isaković, AleksandraCommittee members
Trajković, VladimirPopadić, Dušan
Stanojević, Željka
Volarević, Vladislav
Metadata
Show full item recordAbstract
Eksperimentalni autoimunski encefalomijelitis (EAE) je jedan od
najizučavanijih animalnih modela neuroinflamacije i najčešće je korišćen animalni
model multiple skleroze (MS). Grafenske kvantne tačke (GKT) su tanki listići grafena
(<100 nm), elipsastog oblika, sačinjeni iz 1-10 slojeva grafena. U biološkim sistemima,
GKT mogu da ispoljavaju prooksidativno i antioksidativno dejstvo a u skorašnjim
studijama je pokazano i njihovo imunomodulatorno dejstvo. S toga je cilj ovog
istraživanja je bio da se ispita efekat primene GKT na klinički tok bolesti i inflamaciju
centralnog nervnog sistema (CNS) u EAE modelu kao i celularne i molekularne
mehanizme koji se nalaze u osnovi potencijalnog povoljnog efekta. Efekat GKT kao i
mehanizmi njihovog delovanja su ispitivani primenom in vivo, in vitro i ex vivo
pristupa. Za indukciju EAE korišćeni su pacovi Dark Agouti (DA) soja koji su
imunizovani homogenatom kičmene moždine pacova u kompletnom Frojndovom
adjuvansu. U cilju definisanja faze bolesti u ...kojoj GKT ostvaruju svoje potencijalno
povoljno dejstvo, životinje su tretirane sa GKT (10 mg/kg tt, ip) tokom induktivne,
efektorske ili obe faze bolesti. Ispitivanje lokalizacije GKT u tkivima životinja, analiza
limfocitne infiltracije i apoptoze ćelija CNS i demijelinizacije vršeno je odgovarajućim
histološkim metodama. Koristeći tkiva drenirajućeg limfnog čvora koji oslikava
aktivnost induktivne faze bolesti i kičmene moždine (KM) koja oslikava aktivnost
efektorske faze bolesti, metodom protočne citofluorimetrije je vršena fenotipska
karakterizacija izolovanih mononuklearnih ćelija, qRT-PCR metodom ispitivana
ekspresija gena za medijatore inflamacije a imunoblotom analizirana aktivacija proteina
koji učestvuju u autofagiji i Akt/MAPK signalnim putevima. U in vitro modelu
kokultivacije oligodendrocita i neurona sa stimulisanim limfocitima ispitan je
protektivan efekat GKT tretmana, MTT testom...
Experimental autoimmune encephalomyelitis (EAE) is one of the most studied
animal models of neuroinflammation and also, one of the most commonly used animal
models of multiple sclerosis (MS). Graphene quantum dots (GQD) are thin ellipsoidal
graphene sheets (<100 nm), made of 1-10-layer thick sheets of graphene. GQD exibit
prooxidative and antioxidative effect in biological systems. Also, previous studies show
its immunomodulatory effect. Therefore, the aim of this study was to investigate the
effect of GQD on clinical course of a disease and central nervous system (CNS)
inflammation in EAE model, as well as cellular and molecular mechanisms underlying
its potential protective effect. The effect of GQD and its mechanisms of action were
examined using in vivo, in vitro and ex vivo approaches. Dark Agouti (DA) rats were
immunized with spinal cord homogenate in complete Freund's adjuvant, in order to
induce EAE. GQD treatment (10 mg/kg bw, ip) was administrated during the inductive,
effect...or and both phases of a disease. Using the appropriate histological approaches, the
presence of lymphocyte infiltration, apoptosis and demyelination of CNS was
investigated, as well as the ability of GQD to accumulate in cells of immune system and
CNS. Flow cytometry analysis was used to determine phenotype characterization of
mononuclear cells isolated from the draining lymph nodes (inductive phase) and spinal
cord (SC) tissue (effector phase), while the level of mRNA for the inflammatory
mediators was determined by qRT-PCR. Autophagy and Akt/MAPK signaling
pathways in draining lymph nodes and SC tissue were investigated using immunoblot
assay while the protective effect of GQD treatment on oligodendrocytes and neuronal
cells that were co-cultivated with ConA stimulated lymphocytes in vitro, was verified
by MTT test...