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Samane Adib,Taki Tiraihi,Merzieh Darvishi,Taher Taheri,Hadi Kazemi 한국조직공학과 재생의학회 2015 조직공학과 재생의학 Vol.12 No.1
Human Bone marrow stromal cells (hBMSCs) can differentiate under appropriate experimental conditionsinto neuronal and glial-like cells. This study shows a protocol for producing human neural stem cells (hNSCs)from hBMSCs and the subsequent differentiation of hNSCs into cholinergic neurons (CNs), where sequential mediareplaced the culturing media. hBMSCs have been used in generating cell aggregates (CAs) using bFGF, EGF andB27. The hNSCs were isolated from CAs, and the CNs differentiated from the hNSCs using sequential media, wherebFGF, EGF and B27 were gradually replaced with NGF. The hNSC stemness was checked by RT-PCR of SOX2,Oct-4 and Nanog genes. Fibronectin, CD90, CD106, CD31, nestin, neurofilament 68 (NF-68), NF-200 and ChATimmunostaining evaluated the differentiation of the hBMSCs, the hNSCs and the CNs. FM1-43 was used in studyingthe function of the CNs. The hBMSCs were immunoreactive to fibronectin, CD90 and CD106; they werechecked for lipogenic and osteogenic differentiation. The cells of the CAs were immunoreactive to nestin. ThehNSCs were immunoreactive to nestin and NF-68, also, they expressed SOX2, Oct-4 and nanong. Nestin expressiondeclined sharply following NSC differentiation into CNs, while the expression of NF-200, synapsin I, synaptophysin,MAP-2 and ChAT increased. They were stained with FM1-43, where the synaptic vesicles were releasedfollowing stimulation. The present study demonstrates the conversion of hBMSCs into CASs under appropriate conditions. CAs generated hNSCs, which were induced in order to differentiate into CNs using sequential media, wherethe yield was 83%.