| For near decades,with the development of modern life sciences and technology such as molecular biology and cell biology,research about recovery of spinal cord injury(SCI) have been progressed greatly.Researchers have observed that the self-proliferation,self-restoration and multi-directed differentiation of nervous tissue are very limited after spinal cord injury,just lacking of enough neural stem cells and restriction of micro-environment,no matter what that there are some quiet neural stem cells in the central nervous system of the fetus and adult of the mammalian.Now,it is focused on two aspects such as medication therapy and tissue or cell transplantation. On the data,it could be said that the recovery of structure and function of spinal cord injury can be improved by application of transplanting cell/tissue,neural trophic factors.As we know that neural stem cell(NSC) is kind of pluripotent stem cell,it can be differentiate into neuron,astrocyte and oligodrycyte in vitro and in vivo.NSC can be considered as a good transplantation plant during the therapy of nervous disease,but there need a good marker which can trace the transplantated cells,and green fluorescence protein(GFP) is such a good tracing marker.GFP is a kind of luminescence proteins consisted in coelenterate body such as acaleph,hydra and coral, excited by ultraviolet or blue light,it may eradiate green fluorescence.GFP have a merit such as simple handle and intuitional observation of results compared with other markers.Thereby it is applied extensively in field of gene transfection,fusion of markers,gene therapy,orientation of intracellular protein function and/or its transformation and intracellular tracking of invaded pathogen and etc.Moreover, many studies indicate recently that it has been showed the benign application in many aspects of the tracing,quantitative analysis and measuring of differentiation conditions using GFP in transplanted stem cell and etc.Neuregulin(NRG) is a kind of polypeptides,and activates kinase signal conducting system such as MEK/ERK after it is combined with ErbB receptor.NRG not only takes part in the acetylcholine synthesis of neuro-muscular synapse and the development or growth of Schwann cell and oligodendrocyte,but also the existence and the growth of neuron.The aim of present study was①to isolate and incubate GFP~+-positive neural stem cell derived from fetus spinal cord(NSC) using free-serum cultivation method from embryonic day 16 of GFP~+-pregnancy rat,to observe differentiation function of NSC and expression of Neuregulin-1,simultaneously,and to identify GFP~+-positive neural stem cell with immunofluorescence method.②to establish the rat spinal cord hemi-section model,to verify the existence and differentiation of GFP~+-positive spinal cord-derived neural stem cell after transplanting into the injured models,and then to observe the expression of five antibodies such as Nestin,β-tublin,GFAP,Gal-C and neuregulin-1.③Subsequently,to observe the restoration of neural trace function in experimental animals using BDA neural tracer technique and BBB function score. However,the treatment of rat with spinal cord injury using together application of GFP~+-labeled spinal cord-derived neural stem cell with Neuregulin-1 is not found yet.This study consists of:1 part:material and method:GFP~+-labeled spinal cord-derived neural stem cell was isolated and cultured from the embryonic spinal cord of the GFP~+- pregnancy rat (16d).The expression of Neuregulin-1 and Nestin was observed during the formation of neural sphere.Result:the neural spheres were appeared in 3-rd day after primary incubation,to 7-th day the amounts of neural spheres were increased rapidly,after that, gradually grown up.Among them,biggish spheres may be seen macroscopically as small white granules,and the cells have a close arrangement.During subculture,the differentiation or the death of some NSC may be observed,being adhered to the bottom of cultured dishes.Purified spinal cord-derived NSC could be obtained after subculture repeatedly,and a great deal of clone cells were obtained with consecutive subculture of neural spheres.The neural spheres,formed in 3-rd day after primary culture,had already contained GFP tracer marker.It can be found the existence of a strong green pigment combing with stem cells using fluorescent microscope.For neural spheres whose culture time is 7 days,immunofluorescent combinative staining of Neuregulin-1 and Nestin were performed,indicating a great number of the spheres contain the presence of Nestin and Neuregulin-1.A standard shape of cells were identified by immunofluorescent combinative staining with Nestin,β-tubulin,Gal-C, Neuregulin-1 and GFAP Subsequently,which means that these cells mostly could be differentiated into neuron,astrocyte and oligodendrocyte.conclusion:This study was shown to be successfully isolated GFP~+-labeled spinal cord-derived NSC from GFP~+-labeled pregnancy mouse(16d),processed differentiation function in vitro,to be more easily established the method of neural stem cell's culture.GFP~+-labeled spinal cord-derived NSC may be expressed Neuregulin-1 and Nestin using single immunofluorescence staining.2part:material and method:The rat spinal cord hemi-section model was established, the changes of the cortico-spinal tract were observed using BDA neural tract tracing method.Then GFP~+-labeled spinal cord-derived neural stem cells were transplanted into the sites around injured position,simultaneously with sc injection of Neuregulin-1.After 2 months,it was found that there were expressions of five antibodies such as using immunofluorescence.Result:The rat spinal cord hemi-section model was established successfully,and the perfectibility of SCI was verified using BDA neural tract tracing method.BDA positive staining groups were terminated near injured position.The results of immunofluorescent analysis indicate that GFP~+-labeled spinal cord-derived neural stem cells were still expressed around injured place after 2 months' operation.There are 5 groups of treatment and we found that GFP~+-labeled spinal cord-derived neural stem cell with different dose of Neuregulin-1(20μg/kg) and 100μg/kg subcutaneously respectively,but there was no obvious statistic significance between those two groups, and cells of transplanted DMEM solution group was least.Conclusion:This study was established the rat spinal cord hemi-section model successfully,confirmed perfectibility of model using BDA tracer method.The survived cells of 5 groups of transplanted GFP~+-labeled stem cells with subcutaneously injecting of different dose of Neuregulin-1 into the injured positions after two months' operation got well, proliferated and expressed five antigens.GFP~+-labeled stem cells combing with Neuregulin-1 were more effective than single transplanting GFP~+-labeled stem cell group and the others.3part:material and method:Subsequently,the restoration of neural trace and recovery of the locomotor ability in experimental animals were observed using BDA neural tract tracing method and spinal cord BBB function score.Result:BDA positive staining group was terminated near injured place in the spinal cord hemi-section models.In GFP~+-labeled stem cell group without Neuregulin-1 and GFP~+-labeled stem cell groups with Neuregulin-1,however,the part of the BDA positive-stained cortico-spinal fibres were shown,extending toward the lumbar spine.It was found that the minimal neural fibres extended to near tissues,passing the injured place. There were no significant differences between the all groups(P＞0.05) in a week after operation estimation of the BBB movement score of hindlimb.However there were significant differences between the control group and GFP~+-labeled stem cell group without Neuregulin-1 after two months' injury,and GFP~+-labeled stem cell group without Neuregulin-1 and GFP~+-labeled spinal cord stem cell group with Neuregulin-1(5μg/kg),and Neuregulin-1(20μg/kg) and 100μg/kg(P＜0.05) respectively. Conclusion:It's better for using transplantated GFP~+-positive neural stem cells combing with Neuregulin-1(100μg/kg) and can get higher BBB score although less than standard score,but there is no obvious significance between transplantated stem cells with dose of 20μg/kg and 100μg/kg Neuregulin-1 respectively.
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