Köpek mezenkimal kök hücrelerinin in-vitro nörojenik farklılaştırılması ve özelliklerinin incelenmesi

Bu calismada kopek kemik iligi mezenkimal kok hucrelerin izolasyonu, kulturu,karakterizasyonu ve in-vitro ortamda norojenik nesile farklilastirilmasi incelenmistir.Kopegin iliak krestinden toplanan kopek kemik iligi mezenkimal kok hucreleri (KI-MKH) hucre kulturu ortaminda cogaltildi. Cogaltilan hucrelerin karakterizasyonu icinkoloni olusturma potansiyelleri, empedansa dayali hucre analiz sistemi ile hucrebuyume egrisi, coklu soy ve akim sitometri analizleri gerceklestirildi. Bu asamadansonra kopek KI-MKH’leri, gelistirilen iki-asamali norojenik farklilastirma protokolunetâbi tutuldu. Norokure olusturmak amaciyla yapilan ilk induksiyon asamasinda hucreler;epidermal buyume faktoru (EGF) ve bazik fibroblast buyume faktoru (bFGF) icerennorobazal vasatta kulture alindi. Uc gun suren ilk induksiyon asamasindan sonra eldeedilen norokureler isik mikroskobunda goruntulendi. Norokure olusum asamasindansonra norojenik farklilasmanin ikinci basamagi olan noron-benzeri hucre olusumasamasina gecildi. Dagitilan norokureler, sinir buyume faktoru (NGF), beyin-kaynaklinorotrofik faktoru (BDNF) iceren norobazal vasatta alti gun kulture alindi. Ikinciinduksiyon asamasi sonunda norojenik nesile farklilastirilmis kopek KI-MKH’lerininimmunhistokimya boyamalari yapildi. In-vitro ortamda, kopek kemik iliginden izoleedilen hucrelerin mezenkimal karakterde oldugu, noronal ve glial isaretcileri ifade edenhucrelere farklilasabildigi ve bu hucrelerin ise gelecekte sinir doku hasarlarinintedavisinde fonksiyonel yenilenme icin kullanilabilme potansiyeline sahip oldugugozlendi.AbstractIn this study, we have investigated the mesenchymal properties and neurogenicdifferentiation potentials of cultured canine bone marrow stem cells. Caninemesenchymal stem cells isolated from the iliac crest of canine were proliferated understerile conditions. The mesenchymal properties of the cultured cells were confirmedusing coloniy forming unit, impedance-based cell analysis system, lineage and flowcytometry analysis. At passage 2; BM-MSCs were induced into the neurogenic lineageby a two-step differentiation protocol. Cells were placed in tissue culture flasks coatedwith poly(hydroxyethylmethacrylate) and cultured in the B-27 and N-2 containingneurobasal medium. At the second step, the acquired neurospheres were disaggregatedand the cell suspension was plated in tissue culture flasks which were coated with poly-L-ornithine. The neurosphere-derived cell suspension was then cultured for six days inthe neurobasal medium supplemented with nerve growth factor (NGF) and brainderivedneurotrophic factor (BDNF) for further induction into the neurogenic lineage.For determination of neurogenic differentiation of the mesenchymal stem cells, primaryantibodies were used. The findings indicated that mesenchymal stem cells differentiateinto cells expressing neuronal and glial markers in vitro and some mesenchymal stemcells exhibit neural potential.