The Expression Of Hes1 And Mash1 During The Spinal Cord Stem Cells Specifically Differentiating Into Cholinergic Neurons

Background: The idea that neural stem cells (NSCs) had the ability of self-renewal, proliferation and being induced into neurons, astrocytes and oligodendrocytes, not only broke the theory that the mature central nervous system could not regenerate, but also provided new ideas for the repair of the nervous system damage and and the treatment of degenerative diseases[1]. For the research of NSCs, it was based on maintaining active proliferative capacity of NSCs cultured in vitro. At the same time, a variety of central nervous system diseases were caused by mostly the neuronal lesions of different phenotype and function. However, many studies indicate, before being transplanted into damaged regions, if the neural stem cells were not induced, the specific neurons would be less, and it was difficult that they played their roles. Differentiation of NSCs is a complex process involved many factors. For differentiation conditions and mechanisms of cholinergic neurons in vitro, it has not yet been reached. Objective: By isolation and culture of NSCs, we observe the basic and biological characteristics and understand conditions of proliferation and differentiation of its in vitro. It will lay a foundation for the further study of directed differentiation of NSCs. NSCs are induced to differentiate into cholinergic neurons in vitro, to explore the differentiation conditions and mechanism of NSCs.Methods:The single cell suspensions, derived from rat embryos spinal cord at E17 d, were plated into the serum-free medium. After the neural stem cells clone spheres formed, they were passaged. The neurospheres of third passage were labeled by immunofluorescence detection of Nestin. Then, the spinal cord derived stem cells clone spheres were plated onto cover slips coated by polylysine in 6 well culture-plates. RA, SHH, NT-3 and BDNF as inducing factors were added into the culture medium. Then, the cells were incubated in 5% CO2 humidified atmosphere at 37℃for 14 d. The cholinergic neurons were detected by using immunofluorescence of the choline acetyltransferase (ChAT), which was the distinctive marker for cholinergic neuron.Results: A large number of NSCs were gained which had the ability of self-proliferation, the expression of Nestin, and in vitro maintain stable status for a long time. After being induced one week, the full cell body, thin-multiple branches and strong refraction were presented. 14 d later, a kind of network structure formed among the neural cells. At the moment, immunostaining analyses indicated that 20% differentiated cells were positive for ChAT. Conclusions: The neural stem cells isolated from spinal cord had the ability of proliferating and self-renewing, and possessed ability to differentiate into neurons. When treated with RA, SHH, NT-3 and BDNF, the spinal cord stem cells can be induced to differentiate into cholinergic neurons. Background: The control of growth and development of the neural stem cells (NSCs) involved a number of aspects, including the decision of characteristics, the control of cell number and spatial pattern of cell differentiation. Many gene families were involved in the process. Hes1 and Mash1, which was one of bHLH gene family, as related neural developmental gene, widely expressed in the developing central and peripheral nervous system. Hes genes belong to the negative regulator type bHLH transcription factor. It played an important role in maintaining proliferation of NSCs, making brain cells with the correct number, shape and arrangement. Mash1 gene, as the positive regulator type bHLH transcription factor, promoted neural precursor cells differentiating into neurons. The positive regulator and negative regulator type transcription bHLH transcription factors regulated and controlled each other, which together controlled the differentiation of NSC and determined the direction of their differentiation. In this experiment, by inducing NSCs to differentiate into cholinergic neurons in vitro through adding inducing factors, we observed expression changes of Hes1 and Mash1 gene, in order to know which role they played in differentiating process.Objective: To explore differentiation mechanisms of NSCs, when the embryonic neural stem cells derived from rat spinal cord were induced to differentiate into cholinergic neurons by detecting expression changes of Hes1 and Mash1 gene.Methods: NSCs of the third passage were plated into 6 well cultivate plates coated by poly-L-lysine. They were induced to differentiate into cholinergic neurons by adding RA, SHH, NT-3 and BDNF to medium. The expression changes of Hes1, Mash1 and Nestin gene were analyzed during neural stem cells differentiated into the cholinergic neurons after first week and second week by real-time quantitative PCR.Results:The expression change of Hes1, Mash1 and Nestin mRNA were observed during the progress. The expression of Hes1, Mash1 and Nestin mRNA have decreased in a significant manner after first week, and increased slightly after second week.Conclusion: The Hes1 and Mash1 gene may play an important role in the process of NSCs being induced to differentiate into cholinergic neurons.