The Study On Bone Marrow Stromal Cells Transfected With Brn2, Ascl1 And Mytll Gene Differentiating Into Neuron-like Cells In Vitro

ObjectiveStroke is a devastating disease with loss of neurons and breakdown of reflex arc. It has a high incidence and mortality. The most effective and ideal treatment may be to rebuild the reflex arc, which need the new neurons. The discovery of the neural stem cells in mammals sheds new light on the best recovery from this terrible disease. How-ever, neural stem cells are rare and unaccessible, which has become a most critical problem.Bone marrow stromal cell (BMSC) is a kind of the mesodermal stem cells existing in bone marrow.It is considered as an ideal sources of seed cells with the wide potential of differentiation and the advantage of being isolated and proliferated in vitro easily, which is a prerequisite for the autotransplantation. Up to now, there are many reports on cell's differentiation into neurons by inducer such as retonoic acid,β-mercaptoethanol and baicalin. No study has explore the differention of BMSC induced by Brn2, Ascll and Mytll, three important transcription factor in neurogenesis.Brn2 belongs to a large family of transcription factors that bind to the octameric DNA sequence ATGCAAAT. Brn2 is expressed predominantly in the central nervous system (CNS). It is likely that CNS-specific transcription factors such as these play an important role in mammalian neurogenesis. Ascll gene encodes a member of the basic helix-loop-helix (BHLH) family of transcription factors. This protein plays a role in the neuronal commitment and differentiation and in the generation of olfactory and autonomic neurons. Mutations in this gene may contribute to the congenital central hypoventilation syndrome (CCHS) phenotype in rare cases. Myt11 gene binds specifically to retinoic acid response elements and acts as a transcriptional activator. It may regulate gene expression in the nervous system and pituitary.Therefore, we design to construct an expression vector of Brn2, Ascl1 and Myt11, and explore the differetiation of BMSC transfected with this vector. The result will promote the further research in the biological function of Brn2, Ascl1 and Myt11 gene, the stem cell therapy and gene therapy in cerebral disease.Methods1. After isolated by density gradient contrifugation with Percoll, the rats" bone marrow stromal stem cells were cultured with DMEM containing 20% fetal bovine serum in the plastic flastic. When the cells covered the flask, they were passaged with pancreatin. Their morphology were observed by microscope.2. a 696 bp sequence of Brn2 gene, a 1338bp sequence of Ascl1 gene, and a 3558bp sequence of Myt11 gene were cloned by PCR. The pLV.EX3d.null-CMV>mAscl1/E2A/mBrn2/F2A/mMyt11 plasmid was constructed by Gateway clone technology. Finally,the recombinant plasmid was verified via restrictive endonucleases digest, PCR and DNA sequence analysis.3. The fourth and sixth generation of BMSC were transfected the expression vector with Brn2, Ascl1 and Myt11 by lentivirus.Results1. The vitality of the isolated cells was 99%. After cultured in the plastic flask for 10-14 days, the cells covered the bottom and were passaged. Then they were passaged every 5-7 days. The typical morphology was seen after the cells were passaged for 3-4 times. 2. Analysis of the sequence results showed that the full length of the fragment cloned was correct and the recombinant pLV.EX3d.null-CMV>mAscll/E2A/mBrn2/F2A/mMytll plasmid was successfully constructed.3. The BMSC transfected the vector displayed the neuron morphologies with long and multipolar projections.Conclusions The bone marrow stromal cells transfected with Brn2,Ascl1,Myt11 gene differentiate into neuron-like cells in vitro.