ULOGA AUTOFAGIJE U NEUROTOKSIČNOM DELOVANJU VANĆELIJSKOG ALFA-SINUKLEINA NA SH-SY5Y ĆELIJE HUMANOG NEUROBLASTOMA
Sažetak
Uvod: U osnovi patogenetskog mehanizma Parkinsonove bolesti leži povećano nakupljanje proteina α-sinukleina (ASYN) u dopaminergičkim neuromina substantiae nigrae, što dovodi do, između ostalog, i smrti ćelija. Iako je ranije smatran isključivo unutarćelijskim proteinom, novijim istraživanjima je pokazano da se ASYN nakuplja i u vanćelijskom prostoru. Do nakupljanja ASYN može da dođe usled oštećenja sistema za razgradnju proteina, od kojih je najznačajniji mehanizam autofagija. Autofagija je regulisana produktima ATG gena (engl. autophagy-related genes), a jedan od njih, Atg7, ima bitnu ulogu u formiranju i sazrevanju autofagozoma.
Cilj: Cilj ovog rada je bio da se ispita uloga autofagije u neurotoksičnosti vanćelijskog ASYN na SH-SY5Y ćelije humanog neuroblastoma, diferentovanih u neuronski fenotip.
Materijali i metode: Svi eksperimenti su rađeni na SH-SY5Y ćelijskoj liniji humanog neuroblastoma. Ćelije su diferentovane all-trans retinoičnom kiselinom i tretirane medijumom koji sadrži vanćelijski ASYN. Imunoblot metodom je potvrđeno prisustvo vanćelijskog ASYN i praćena promena eskpresije markera autofagije, proteina LC3-II i beklin-1. Metodom transfekcije sa malom interferirajućom RNK je inhibirana ekspresija Atg7 proteina. Vijabilitet i broj ćelija je određen kristal violet testom.
Rezultati: Vanćelijski ASYN dovodi do značajnog smanjenja vijabiliteta SH-SY5Y ćelija, što je praćeno povećanjem nivoa markera autofagije, proteina LC3-II i beklin-1. Analizom ćelijskog vijabiliteta primećen je značajan pad u broju živih ćelija kod kojih je utišan gen za Atg7, a gajene su u prisustvu vanćelijskog ASYN.
Zaključak: Vanćelijski ASYN dovodi do smanjenog preživljavanja SH-SY5Y ćelija diferentovanih u neuronski fenotip, što je praćeno indukcijom autofagije. Inhibicija autofagije preko utišavanja Atg7 gena dovela je do povećane osetljivosti ćelija gajenih u prisustvu vanćelijskog ASYN, što ukazuje na moguću protektivnu ulogu autofagije u neurotoksičnosti izazvanoj nagomilavanjem ASYN u vanćelijskom prostoru.
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