The Relationship Between NRSE/RE-1 And Induced Differentiation From Mouse Embryonic Stem Cells To Neural Cells

The relationship betweeen NRSE/RE-1 and induced differentiation from mouse embryonic stem cells to neural cellsintroductionHuman health has been acutely threated by Nervous system diseases,which of them such as Pakinson's disease,Alzheimer disease,spinal cord injury, are result of that the injured mature neural cells can be recruited by division growth of neural cells. The stem cells are derived on inner cell mass, which have the pluripotency of self-duplication and be induced to three germ-layers. Lots of research indicate that they can be differentiated to neural cells to be graft to recover the damage of neural system. But we have not understood mechanism of differentiation of neural cells.The expression product of REST／NRSF which inhibit the expression of neural differentiation genes can bind NRSE/RE-1, the promote district of neural differentiation genes. Expression of RESE/NRSF is obviously downregulated in the natural differentiated neural cells in vivo, so we consider them to be negative regulation genes of neural differentiation.We have make use of sequential neural induction which is builded all by ourselves, induce embryonic stem cells to neuron, and initially investigate the mechanism of neuron induced from embryonic stem cells:1,The result of genetic expression microarray confirm that the expression of REST/NRSF obviously reduce in the process of ESCs induced to neural cells, but the expression of including NRSE/RE-1 of neural terminal differentiation gens that be affected by its protein generally advance.2,Used RNAi directly interfere REST/NRSF binding with neural differentiation genes, promote neural genes expression, induce mESCs to neural cells.We have chosen some genes which may have NRSE/RE-1, such as P2ry12, Nptxr, Syp, BDNF, Syt2, Syt7,Ntrk3,Cdh4, Cacna2d3, Atp2b2,Rgs7. So my major work is using real-time PCR to precisely quantitative assay the expression of those genes in the course of differentiation by sequential induction in vitro, and investigate the mechanism of action of them, and approach the master genes about neural cell differentiation, and possible differentiation marker genes.Method1,Culture and induction of ESCsESCs line(AB2.1) were routinely propagated on feeder cells. To initiate induction, undifferentiated ESCs were dissociated and replanted on none-coated tissue culture plastic dishes in NSC medium. The medium was changed every other day into serum-free NSC medium containing bFGF. On the fourth day, The medium was changed to bFGF free N2-B27 medium.2,Identification of the undifferentiated ESCs and neural induced cells.(1) Alkaline phosphatase test of undifferentiated ESCs(2) Immunostaining of neural specific antigens: nestin,NCAM1,NSE,NeuN and GFAP.(3) NCAM1,DAPI counterstain to determine ratio of differentiation3,Sieve master genes on differentiation processGene expression of undifferentiation and on the fourth,eighth day of screened genes identification by real-time PCR analysis, and analysis variation rule.Results1,ESCs can be induced into neuron-like cells by sequential neural inductionThe undifferentiated ESCs grew in colonies, with clear bound. Alkaline phosphatase staining showed dark brown positive particles were distributed in every ESC colony. After the sequential neural induction, the cells converted into neuron-like cells, with homogeneous forms, which had round bright cell bodies and thin long bipolar or multipolar processes.2,The differentiated cells on the tenth day were immunopositive for NCAM1 and 86.2％±5.5％induced cells were NSE-positive.3,The result of real-time PCR analysis is that to follow with differentiation period, 11 kinds of genes, containing NRSE/RE-1, expression increase.Conclusion1,The sequential neural induction can convert ESCs into neurons effectively. The ratio of induced differentiation is 86.2％±5.5％2,Those 11 genes containing NRSE/RE-1 are up-regulation which have relationship with differentiation of ESCs to mature neurons, and they possibly be impled master genes about neural cell differentiation.3,The genes of Cacna2d3, Nptxr, P2ry12, Rgs7 are to be the reference marker genes in the course of ESCs induced to neural cells.