| Objective The research of biological characteristics of NSCs derived from iPS cells,and discussing the effects of Wnt/?-catenin signaling pathway on transcription factor TCF1 and LEF1,Ngn1 and Ngn 2 during the neural differentiation of iPS cells.Methods(1)The effect of down-regulating the Wnt/?-catenin signaling pathway on proliferation and migration of iPS-NSCs in vitro:(1)MEF was prepared.(2)Cultured and induced the mouse iPS cells into NSCs by N2B27 combined with RA in vitro and identified by immunofluorescence.(3)During the differentiation of iPS cells into NSCs,the cells were divided into normal group and DKK-1 group,which was down-regulated the Wnt/?-catenin signaling pathway.(4)The CCK-8 method was used to detect the growth curve of iPS-NSCs,and research the effect of down-regulation of Wnt/?-catenin signaling pathway on proliferation of iPS-NSCs.(5)The transwell method was used to detect the migration ability of iPS-NSCs,and study the effect of down-regulation of Wnt/?-catenin signaling pathway on iPS-NSCs migration.(2)The effect of down-regulation of Wnt/?-catenin signaling pathway on differentiation of NSCs derived from iPS in vitro:(1)Cultured and induced the iPS-NSC differentiate into neural cells;(2)The differentiation ability of iPS-NSCs into neurons and astrocytes was identified by immunofluorescence;(3)The flow cytometry was adopted to study the effect of down-regulation the Wnt/?-catenin signaling pathway on apoptosis of iPS-NSCs.(3)The effect of down-regulation the Wnt/?-catenin signaling pathway on the expression of TCF1?LEF1,Ngn1,Ngn2 and GSK3? during the differentiation of iPS cells into NSCs:(1)The western-blot and q RT-PCR were used to detect the expression of transcription factors TCF1?LEF1,Ngn1,Ngn2 and GSK3? in Wnt/?-catenin signaling pathway during the period of differentiation of iPS cells into NSCs;(2)The small molecule inhibitor DKK-1 was taken to down-regulate the Wnt/?-catenin signaling pathway,and the western-blot and the q RT-PCR were used to detect the expression of transcription factors TCF1?LEF1,Ngn1,Ngn2 and GSK3? in Wnt/?-catenin signaling pathway in the course of differentiation of iPS cells into NSCs..Results(1)The effect of down-regulation of Wnt/?-catenin signaling pathway on proliferation and migration of iPS-NSCs in vitro:(1)The MEF was fibrous and fusiform,and was easy to adhere to the wall,growing quickly.(2)The iPS cells exhibited clonal-generating capability,and developed embryoid with tridermal structureand,Nestin and Sox2 were expressed positivly.(3)The growth curve of Wnt/?-catenin was "S" shape,which was no significant change after down-regulation of Wnt/?-catenin signaling pathway(P>0.05).(4)iPS-NSCs had the ability to migrate in vitro,which was downfall after down-regulation Wnt/?-catenin signaling pathway(P<0.05).(2)The effect of down-regulation of Wnt/?-catenin signaling pathway on differentiation of NSCs derived from iPS in vitro:(1)Immunofluorescence assay showed that some cells were positive for early neuron-specific marker ?III-tubulin and astrocyte surface specific marker GFAP.(2)During the differentiation of iPS-NSCs into neural cells,apoptosis occurred in the cells,which could decrease after down-regulation the Wnt/?-catenin signaling pathway(P<0.05).(3)The effect of down-regulation the Wnt/?-catenin signaling pathway on the expression of TCF1?LEF1,Ngn1,Ngn2 and GSK3? during the differentiation of iPS cells into NSCs:(1)The expression of TCF1 and LEF1 decreased first and then increased(P<0.05),which were all higher than those in normal group after down-regulated the Wnt/?-catenin signaling pathway.(2)The expressiones of Ngn1 and Ngn2 were all increased on the sixth day of NSC(P<0.05),which were all higher than those in normal group after down-regulated the Wnt/?-catenin signaling pathway(P<0.05).(3)The expression of GSK3? were decreased in the Wnt/?-catenin signaling pathway during the differentiation of iPS cells into NSCs(P<0.05).which was increased in the later stages of neural differentiation after down-regulation of Wnt/?-catenin signaling pathway(P<0.05).Conlusion(1)iPS cells have the ability of proliferation,migration and to differentiate into neuronal cells and astrocytes.(2)When the Wnt/?-catenin signaling pathway was down-regulated,it had no effect on proliferation,the ability of migration was decreased and the iPS-NSCs was promoted to differentiate into neuronal cells and astrocytes,the apoptosis rate of iPS-NSCs was reduced.(3)In the period of differentiation of iPS cells into neural cells,the expression of transcription factors such as TCF1,LEF1,Ngn1,Ngn2 and GSK3? were promoted after down-regulating the Wnt/?-catenin signaling pathway.The results shown that the Wnt/?-catenin signaling pathway was related with the progress of iPSCs differentiated into neural stem cells rely on the transcription factors includes TCF1,LEF1,Ngn1 and Ngn2. |
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