Proliferation,Growth And Differentiation Of Olfactory Ensheathing Cells And Sodium Nitroprussid On Spinal Neural Stem Cells

Recently,there is great improvement on the research of regeneration after spinal cord injury.Cells transplanting is thought of one of the bright ways.Neural stem cells (NSCs) can be differentiated into neurons to provide missing cells as seed cells.Olfactory ensheathing cells (OECs) can supply many neurotrophic factors,cell matrix and nerve cell adhesive molecules.OECs can make neuron survive and axon regenerate.Another aspect OECs can form natural integration with central nervous system (CNS).The most important aspect is OECs can parcel the regenerate axon and follow it to target cell, avoiding to contact with inhibitor.In these researches we want to know: how to make spinal NSCs differentiate into neurons; if OECs can make NSCs survive and differentiate into neurons.There are two parts in these studies.The first part is to investigate inhibitory effects of sodium nitroprusside (SNP) on the proliferation and the differentiation of spinal neural stem cells into neural cells in vitro. Spinal neural stem cells were cultured and purified on the media without serum, with different concentration of sodium nitroprusside.The quantitative and morphological changes and the expression of MAP2, GFAP and MAG were observed and photomicrographed by phase contrast microscope.We found theamount of living cells in experiment groups was less than in the control group (5.1+1.27)xl05/m,(3.3+0.83)xl05/Ml,(2.2+0.60)xl05/mL and (1.4+0.32) x105/mLP<0.05. The majority of spinal neural stem cells protruded with several long processes and expressed high level of MAP2, while minority of spinal neural stem cells protruded with several short processes which were GFAP positive.The remaining small part protruded with several crassitude processes which were MAG positive. Ratio of neuron is different(30.2+3.8)% (50.9+2.8)% (58.6+3.3)% and (66.7+4.1)%)(P<0.01). Conclusions is Sodium nitroprusside can inhibit the proliferation and promote the differentiation of spinal neural stem cells into neuronal but not glial cells in vitro.The second part is to investigate the function of olfactory ensheathing cells (OECs) on spinal neural stem cells (NSCs) in vitro.The sample was divided into six parts. In group one, NSCs were cultured in DMEM-F12 (DF12) in which OECs had grown for one day.In group two, OECs and NSCs were co-cultured.In group three NSCs were cultured in DF12 without serum. In group four, NSCs were cultured in DF12 with serum.In group five, OECs and NSCs were co-cultured in DF12 without serum.In group six, OECs provided conditioned medium.The morphological changes and the expression of MAP2, GFAP and MAG were observed and photomicrographed by phase contrast microscope.Result is, in group one, the majority of spinal NSCs expressed high level of MAP2, while minority of spinal neural stem cells were GFAP positive.The remaining small part was MAG positive,While in group two, five, both growth and differentiation of NSCs were inhibited in place where OECs were eugenic.But NSCs grew normally and performed like those in group one,And NSCs grew and differentiated around OECs.There are plenty of MAP2- positive cell in the place NSCs differentiated. In group three and four The majority of spinal NSCs expressed GFAP, small part were MAP2 positive.In group five, the morphological of OECs changed.Without serum OECs turned to dipolar or multipolar.We conclude that OECs and theirsecretion can support growth of NSCs and make them differentiate into neurons.