| Research background and purpose:Nestin expresses in neuroepithelial cells,neural stem cells(NSCs)and astrocytes(AS)in the development of the central nervous system(CNS).Neural stem cells are subpopulations of cells with self-renewal and multi-directional differentiation potential,capable of differentiating into neurons(neurons),astrocytes and oligodendrocytes.Published studies have shown that during the development of the nervous system,the expression of Nestin is dynamically changed:at the embryonic stage,the neural precursor cells temporarily express Nestin,and later gradually it was replaced by glial fibrillary acidic protein(GFAP,a marker of astrocytes),β-tubulin-Ⅲ(β-3-tublin,neuron marker).Nestin always has long been regarded as a marker of neural stem cells.Recently,more and more studies have shown that Nestin is not only a cell marker,but also a functional protein.Based on the background,this study proposes the hypothesis that Nestin affects the proliferation and differentiation of neural stem cells and astrocytes.We detemined the Nestin expression pattern in mouse brain;Then,we changed the expression of Nestin protein through gene transduction and gene overexpression or knockdown to study its function in NSCs and astrocytes,and to deepen the understanding of Nestin protein.Through its function on neural stem cells and astrocytes,it proves that it has potential to repair nerve damage and treat neurodegenerative diseases.Methods:1.Whole brain sections of wildtype(WT)C57 mice at different time points in embryonic and after birth were collected for immunohistochemistry,and the expression patterns of Nestin were observed.2.We establish a culture system of NSCs in vitro.Immunofluorescence was used to detect the purity of neural stem cells after adherence and immunofluorescence staining using neural stem cell marker Nestin,astrocyte marker S100,neuronal marker Tuj1,oligodendrocyte marker O4 Pure neural stem cells were abtained with FACs on the CD 133 positive expression,ACSA2(astrocyte marker),PSA-NCAM(neuron)and O4 negative expression.3.Detecting whether primary stem cells have the potential for proliferation and differentiation.The neural stem cells were isolated from the brains of P1(postnatal 1)and P2 mice,cultured in vitro,and formed neurosphere;then digested into single cells and adherently cultured with addition of serum to undergo differentiation for 3-5 days,by using immunofluorescence method,differentiation into astrocytes(S100+),neurons(β-3-tublin/Tuj1+)and oligodendrocytes(O4+).4.Nestin knocked down and overexpression plasmids were constructed for lentiviral packaging,and nestin in neural stem cells was knocked down or overexpressed by lentivirus infection.The proliferation of NSCs was detected by immunofluorescence(Ki67)and the direction of differentiation by Tuj1/S100.5.Astrocytes were isolated from the cerebellum of WT mice with P6,P7 or P8 days,detected their purity(S100+)and proliferation in vitro by immunofluorescence,Astrocytes were knocked down Nestin by lentiviral infection,and detected the effect on proliferation by immunofluorescence..6.Exploring the causes of astrocyte proliferation,detected if the Sonic hedgehog(Shh)signaling pathway affect its proliferation by immunofluorescence.At the same time,the brain slices of P8-day ptchl-lacz mice were taken,and the expression of lacz gene was detected by β-gal(β-galactosidase)activity to detect whether ptchl was expressed,that is,whether the Shh signaling pathway was activated or not,and the body was also detected.7.Collection of astrocyte from normal and Nestin-deficient mouse cerebellum for in vitro culture,and add Shh protein for treatment.The expression and proliferation of Nestin protein were detected by immunofluorescence.Results:1.The results of immunohistochemistry showed that Nestin protein was widely distributed in the molecular layer of cerebellum,cerebral ventricle and hippocampus of mice,mainly expressed in neural stem cells and astrocytes.The structure of Nestin positive expression was mostly the slender filamentous fibers are densely distributed and relatively evenly distributed.2.The primary extracted NSCs were able to self-renew in vitro to form neurospheres and,in the case of serum-induced 72-hour differentiation,the results of immunofluorescence showed that NSCs differentiated into 74%of astrocytes(S100),21%.Neurons(Tuj 1)and 5%oligodendrocytes(04).3.Detect the purity of neural stem cells after adherence by IF,that is,the ratio of Nestin+,S100-,Tuj1-cells was 37%,and the purity of suspended neural stem cells was detected by flow cytometry,namely CD133+,ACSA2-,O4-,The proportion of cells in PSA-NCAM-was 32.3%,and the difference between the two was not large.It proved that the purity of neural stem cells extracted in vitro was about 30%,and the purity was not high.4.Lentivirus infection in adherent NSCs,knocked down Nestin protein,and then NSCs proliferation slowed down,while serum-induced NSCs differentiation,NSCs differentiation to neurons increased by 30.5%after Nestin knocked down.In contrast,when the Nestin protein was overexpressed,the proportion of differentiation into neurons is significantly reduced.5.The purity of primary astrocytes extracted by immunofluorescence was over 90%(S100+),the proliferation level was about 20%,and Astrocyte proliferation decreased after Nestin knocked down.6.Further research findings,the proliferation in vitro and in vivo depended on Shh signaling pathway.When the pathway was inhibited,the proliferation level significant decline.7.After Nestin knocked down,the ability of AS to respond to the shh signaling pathway is reduced,thus resulting in decreased proliferation by IF detected.Conclusion:Nestin protein is widely distributed in the brain of young mice and is highly expressed in neural stem cells and astrocytes.The protein level of Nestin affects the development of both,proliferative ability and differentiation direction of stem cells.When the level of Nestin of neural stem cells decreases,the proliferative capacity is significantly reduced,and it induces its directed differentiation into neurons.When a constant expression of Nestin is given to stem cells,its differentiation is also affected,and it almost no longer differentiates into neurons.For astrocytes,the proliferation of both in vitro and in vivo depends on the Shh signaling pathway,and affects its ability to respond to Shh protein and leads to a significant decrease in proliferation after Nestin knocked down.This study aimed to learn the specific functions of Nestin protein in neural stem cells and astrocytes,and found that it has a role in proliferation and differentiation,which may be a therapeutic role in central nervous system injury and degenerative diseases. |
