| Backgroud: Adult neurogenesis is important for the development of regenerative therapies for human diseases of the central nervous system(CNS)through the recruitment of adult neural stem cells(NSCs).NSCs are characterized by the capacity to generate neurons,astrocytes,and oligodendrocytes.To identify key factors involved in manipulating the adult NSC neurogenic fate thus has crucial implications for the clinical application.Here,we report that BAF45 D is expressed in the central canal(CC)of the adult spinal cord.Coexpression of BAF45 D with glial fibrillary acidic protein(GFAP),a radial glial like cell marker protein,was identified in the the adult spinal cord CC.Quantitative analysis data indicate that BAF45 D is preferentially expressed in the neurons of the adult CNS.Furthermore,during the neuroectoderm differentiation of P19 cells,BAF45 D is required for the expression of PAX6,a neuroectoderm determinant that is also known to regulate the self-renewal and neuronal fate specification of adult neural stem/progenitor cells.Together,our results may shed new light on the expression of BAF45 D in the adult neurogenic zones and the contribution of BAF45 D to early neural development.Objective: Main research aspects: 1): expression of BAF45 D in adult neurogenic area;2): co-expression of BAF45 D and NEUN,BAF45 D and GFAP,BAF45 D and BETA-III-tubulin in adult rat spinal cord;3): to investigate the role of BAF45 D protein in the differentiation of P19 embryonic cancer cells into neuron-like cells induced by retinoic acid(RA)in vitro;Methods: 1):spinal cord sections were prepared from C57/BL6 adult rats(10-12 weeks,body weight 25-30g),and the expressions of BAF45 D,NEUN,GFAP,BETA-III-tubulin in the spinal cord were detected by immunohistochemistry(IHC)and immunofluorescence(IF).2):retinoic acid(RA)was used to induce the neuronal differentiation of rat P19 cells,and the expression of BAF45 D,OCT4,NESTIN and PAX6 were detected by western blotting(IB).3): protein suspensions from rat spinal cord and hippocampus were prepared and the expressions of BAF45 D,NEUN,GFAP and GAPDH were detected by western blotting(IB).Results: 1):BAF45D was positively expressed in the non-ependymal nucleus and central ependymal nucleus of the spinal cord of adult rats.2):GFAP was strongly expressed in cell processes,and BAF45 D and GFAP could be co-expressed in the same cell.3):BAF45D can be expressed in neurons marked by NEUN and BETA-III-tubulin,and BETA-III-tubulin with BAF45 D are co-expressed in the cytoplasm;4):GFAP was expressed in both gray matter and white matter of spinal cord,while BAF45 D with BETA-III-tubulin were mainly expressed in gray matter.5): on the fifth day after RA induced P19 cells,cell morphology was observed under light microscope,and most of the protuberant structures were observed.P19 cells without RA induction maintained the original cell morphology.6): during RA induction,the expression of Nestin,a specific marker of neural differentiation,increased gradually with the extension of induction time,indicating that RA can effectively stimulate P19 cells to differentiate into nerve cells.7): in the process of RA inducing P19 neural differentiation,Pax6 presented a gradually increasing trend in the early stage,reached a peak on the fourth day,and then gradually decreased in expression;The expression level of OCT4 decreased gradually in a time-dependent manner.The expression of BAF45 D increased gradually and presented a time-dependent relationship.8): the expression of GFAP in the spinal cord was significantly higher than that in the hippocampus.BAF45 D and NEUN were slightly more expressed in hippocampus than in spinal cord.Conclusion: 1):BAF45D was expressed in ependymal cells of the central spinal canal of adult rats.2): BAF45 D is preferentially expressed in mature neurons in adult rat spinal cord;3): retinoic acid(RA)can successfully induce P19 cells to differentiate in the direction of nerve,and the production of PAX6 in the differentiation process is related to BAF45 D to some extent;... |
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