Osteogena diferencijacija mezenhimskih matičnih stanica pomoću adenovirusnog prijenosa BMP-2 gena

master's thesis

• Title (english): Osteogenic differentiation of mesenchymal stem cells by adenovirus mediated BMP-2 gene transfer
• Author: Mirta Matišić
• Mentor: Inga Marijanović (mentor)
• Committee member: Maja Matulić (predsjednik povjerenstva)
• Committee member: Inga Marijanović (član povjerenstva)
• Committee member: Ana Galov (član povjerenstva)
• Committee member: Sandra Radić Brkanac (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2013-02-11, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Mezenhimske matične stanice (hMSCs, prema engl. human mesenchymal stem cells) predstavljaju jedinstvenu populaciju somatskih matičnih stanica koje se mogu izolirati iz različitih vezivnih tkiva i organa u odraslom organizmu, a prvotno su dobivene iz koštane srži. S obzirom na svoju multipotentnost, odnosno veliku sposobnost diferencijacije u različita tkiva mezenhimskog podrijetla kao što su kost, hrskavica, mišićno i masno tkivo, te zbog jednostavne izolacije i brzog in vitro klonskog umnažanja, hMSCs se koriste u raznim istraživanjima u području regenerativne medicine. Da bi se usmjerile prema osteogenoj diferencijaciji potreban je određeni biološki signal koji će ih potaknuti na diferencijaciju. Takav osteoinduktivni signal posjeduje koštani morfogenetski protein 2 (BMP-2, prema engl. bone morphogenetic protein 2). Kako bismo dostavili gen od interesa u mezenhimske matične stanice, koristili smo replikacijski defektne adenovirusne vektore serotipa 5 (RdAd5) kojima nedostaje E1-područje virusnog genoma. To im omogućava replikaciju samo u stanicama koje imaju stabilno ugrađeno E1-područje u svom genomu, stoga su za umnažanje adenovirusnih vektora korištene AD-293 stanice. Istraživanje obuhvaća pokuse koje možemo podijeliti u dvije skupine: prvi dio se odnosi na umnažanje, pročišćavanje i određivanje koncentracije infektivnih adenovirusa, dok se drugi dio odnosi na sam proces transdukcije mezenhimskih matičnih stanica, te potvrdu osteogene diferencijacije pomoću citokemijskog bojanja i specifičnih osteogenih biljega. Glavni ciljevi ovog istraživanja su: uzgoj virusa i određivanje virusnog titra, vizualizacija i određivanje razine transdukcije, te potvrda osteogene diferencijacije mezenhimskih matičnih stanica.

Abstract (english)

Human mesenchymal stem cells (hMSCs) are a unique population of adult stem cells which can be found in different organs and tissues in an adult organism, but they are originally isolated from bone marrow. Being multipotent, the hMSCs have a great capacity of differentiation in different tissues of mesenchymal origin, including bone, cartilage, muscle and adipose tissue. The hMSCs are greatly used in research of regenative medicine because of their simple isolation, fast multiplication in vitro and multipotency. In order to direct the hMSCs towards the osteogenic differentiation, a specific biological signal is needed to induce the differentiation. Such an osteoinductive signal is the bone morfogenetic protein 2 (BMP-2). Replication-defective adenovirus serotype 5 vector (RdAd5) which exhibits deletions in the E1 region, was used for gene transfer. Adenoviruses RdAd5 were multiplified in AD-293 cells, because they are able to replicate only in certain cells which have integrated E1 region. Research includes experiments which can be divided into two groups: the first group refers to multiplication, purification and determination of viral concentration and another one is associated with transduction of mesenchymal stem cells, osteogenic confirmation which is based on cytochemical staining and specific osteogenic markers. The main aims of this study are: virus cultivation and determination of virus titer, visualisation and determination of transduction level and confirmation of osteogenesis of mesenchymal stem cells.

• Keywords: mezenhimske matične stanice; adenovirusi; BMP-2; osteogeneza
• Keywords (english): mesenchymal stem cells; adenoviruses; BMP-2; osteogenesis
• Language: croatian
• URN:NBN: urn:nbn:hr:217:061130
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-04-27 10:31:22