Effect Of Sonic Hedgehog On The Proliferation And Differentiation Of Neural Stem Cells

Neural stem cells(NSCs) are multipotential cells that have the capacity to proliferate in an undifferentiated state for self-renew,and to give rise to neurons and neuroglial cells.In nervous system development process,the majority of neural stem cells differentiate into glial cells and only a small number of them differentiate into neurons.Lot of studies in vitro or in vivo also showed that,in the absence of special intervention,the majority of neural stem cells will differentiate into astrocytes,the products of neuron or oligodendrocyte were very few.However,the main purpose of neural stem cells transplantation is to replace the damaged/disappeared neurons or oligodendrocytes.So,how to promote neural stem cells into neurons or oligodendrocytes is a very important in the neural stem cells research.Sonic Hedgehog(SHH) signaling controls many aspects of ontogeny, orchestrating congruent growth and patterning.Based on the mechanism of SHH regulates brain development and the progress of the current studies,we assume that SHH may affect the proliferation and differentiation of neural stem cells,but so far, there is no relevant report.So,the present study is proposed to explore the effects of SHH on the proliferation and differentiation of neural stem cells in vivo,and we hope to provide a basis for future studies.The subject is composed of two parts as following.PartⅠ Objective:To isolate the neural stem cells from the brain of embryonic rat,and to identify the property of neural stem cells by detecting specific protein expression as well as the capacities of proliferation and differentiation.Methods:The neural stem cells isolated from the brain of embryos(E9.5d) were cultured in the serum-free culture medium containing basic fibroblast growth factor and epidermal growth factor.The passaged cells were utilized to detect the expression of neural stem cell-specific marker nestin by fluorescence immunocytochemistry;to detect the BrdU positive cells 3h after the cells be treated with BrdU;to do theβⅢ-tubulin,GFAP,Rip and nestin fluorescence immunocytochemical detection for cell differentiation 7d after the cells be treated with the medium containing serum but not growth factor.Results:The neural stem cells could grow quickly and form neurospheres in culture, and the cells in the neurospheres were positive for nestin which is the neural stem cell-specific marker;the obtained cells could combined BrdU into the nuclei,which means it could proliferate;and it could differentiate into neurons,astrocytes and oligodendrocytes.Conclusion:We had obtained the neural stem cells with their characteristics,which are the expression of specific markers,and the abilities of proliferation and differentiation.PartⅡObjective:To explore the sonic hedgehog(SHH) on the proliferation and differentiation of neural stem cell.Methods:After be treated by sonic hedgehog(SHH),retinoic acid(RA) and PBS respectively,the properties of proliferation(percentage of BrdU-positive cell) and differentiation(percentage ofβⅢ-tubulin,GFAP,Rip,and nestin-positive cells) were detected on the passaged neural stem cells by immunocytochemistry.Results:The BrdU,βⅢ-tubulin and Rip-positive rate were significantly higher in the SHH group than that of the RA group and PBS group.The nestin-positive rate was significantly lower in the SHH group than of the RA and PBS groups.And there was not significant difference in GFAP and nestin-positive rate among the three groups.Conclusion:SHH can enhance neural stem cell proliferation capacity,and promote it to differentiate into neuron and oligodendrocyte.