| BackgroudSpinal cord injury (SCI) is serious clinical problem, because it invariably results in the loss of functional impairment, paraplegia or tetraplegia. However the functional recovery following SCI is very poor due to the neural lost and limited axonal regeneration. To data, there was no effective treatment. There are two sequent processes of SCI, including primary and secondary injury. It can lead to apoptosis and necrosis of neural and glia cells, demyelination of axon. The pathological change of SCI is mainly hemorrhage of lesions due to primary injury, including apoptosis and necrosis of neural cells and angiorhagia of spinal cord, especially the obvious spinal cord cenereal hemorrhage followed by secondary injury in sequence. It relates to many mechanisms, including ischemic lesion of spinal cord injury, energy metabolic disturbance of nerve tissue, excitatory amino acid toxicity effect, calcium overload of neural cells, cytokine and apoptosis, effect of free radical and lipid peroxidatic reaction, neurotoxic effect nitrogen monoxidum, and so on. It would become capsular space of cellular necrosis, spinal cord tissue lenition and glial scar in final. Neurological function of spinal cord would be lost permanently. So how to substitute the lost neural cells of primary spinal cord injury and improve the adverse micro-surrounding caused by secondary spinal cord damage will be a important topic of treating the spinal cord injury.Stem cells substitution treatment of spinal cord injury is a new way. To repair the spinal cord injury, it need generate the neuron and axon, intergrate the neuron and reconstruct the synaptic. So we must repair the motor and sensory neuro-fiber, resubstitute the lost neuron and glia cells with normal neural cells. It may achieve fuctional recovery through the following mechanism, including reconstruction of neural connection with new neural cells, providing suitable surrounding for regeneration of neuron and axon, some secretary nutrition factor improving microenvironment of spinal cord lesion, and so on.Stem cells for the transplantation must have the following abilities, easily obtained, quick multipliced in vitro, weak immune reaction, long-term surviving in nevous tissue of the host. The following stem cells could be the seeds of transplantation, brain derived neural stem cells, schwann cells, olfactory ensheathing cells, embryonic stem cells, umbilical cord blood cells, bone marrow stromal cells. Neural stem cells have the multiple potentiality of self-renew and differentiating the neuron, astrocyte, oligodendrocyte. It showed that there were hematopoietic stem cells and bone marrow stromal cells in bone marrow, and BMSCs can differentiate into neuron and glial cells in certain surrounding. BMSCs have the better merit than other stem cells such as brain derived stem cells, embryonic cells, umbilical blood cells, and so on. It can be obtained easily from the body of adult and have little damage to him. Moreover the self-transplantation can overcome the ethical and immunological problem of using foetus tissue. So transplantation of BMSCs for spinal cord injury had been the hot topic.But the adverse micro-surrounding of spinal cord injury may result to damage of implanting stem cells and lead to the apoptosis and necrosis of them. How to improve the adverse micro-surrounding and the survival rate of implanting stem cells. Some researcher found that Granulocyte colony-stimulating factor (G-CSF) can participate the compensation and protection of neural tissue injury and lighten the extent of neural lesion. Moreover it can protect apoptosis and necrosis of the neural cells in the spinal cord lesion. And it can mediate the growth and differentiation to hematopoietic cells and have the biological ability of stimulating bone marrow cells multiplication and maturate. Also it can mobilize a large mounts of BMSCs into blood circulation and make them migrated to the lesion of spinal cord. Some researchers reported that G-CSF can obviously improve the expression of nestin protein around the brain injury lesion of experimental animals and lessen the death rate of them and the lever of neural tissue damage. Others suggest that G-CSF have the ability of inducing endothelial cells migration and multiplication, and improve the vascular formation of injury lesion, besides the stimulating haematogenesis. It can migrate the BMSCs in blood circulation to the part of the spinal cord injury and differentiate into neurons and glias, and release some kind of neurotrophic factors. It may repair the neural tissue through the anti-apoptosis, anti-inflammatory, improving vascular formation, and differentiating neural stem cells.So we want to obtain a large number of purified BMSCs through cultivating and amplificating in vitro, and simulating the surrounding of cells apoptosis and necrosis in spinal cord injury by co-cultivate G-CSF and BMSCs, to observe the effect of G-CSF by checking the cells survival rate of co-cultivated BMSCs and glutamate. Moreover, we establish the stable model of the spinal cord transection in rats and want to obtain the therapeutic effect of combination of BMSCs transplantation and G-CSF treatment by observing the motor function of animals, histological change, cells apoptosis of spinal cord lesion.Objective1. To explore the effects of glutamate to the rats BMSCs survival rate by co-cultivated the with different concentration glutamate.2. To explore the effects of G-CSF by co-cultivated the rats BMSCs with G-CSF of different concentration followed by certain concentation glutamate.3. To explore the therapeutic effect of combination of BMSCs transplantation with G-CSF for the spinal cord transection of rats.Chapter I Cultured and identification of BMSCs and the effect of glutamate to BMSCs survival rate of ratsObjective:To establish the cultivating and amplificating methods of the rats BMSCs in vitro, and identify the obtained cells by obversing cells appearance, phaenotype character and expression of mark protein. To observe the effect of glutamate to BMSCs survival rate of rats.Methods:Bone marrow stromal cells were cultured in vitro for purification and amplification by application of density gradient centrifugation from the rat femurs and tibias. Bone marrow stromal cells were observed for the morphological characteristics under phase-contrast microscopy; The surface protein expression of BMSCs was observed by immunohistochemistry under fluorescence fluorescence microscope, and their surface markers were cheched with the application of flow cytometr; group A was control, which was BMSCs without glutamate, B,C,D were respectively BMSCs co-cultured with 10,30,50mmol/l glutamate for 24h. It was observed and recorded that the numbers of normal and abnormal cells from 50 cells by fluorescence microscopy through the application 10,30,50 mmol/1 glutamate and BMSC co-cultured for 24h, contrast to group A, with the Hoechst33342 fluorescence staining. The cell survival rate were recorded and analysied with one-way ANOVA in each group. LSD was used when homogeneity of variance was assumed and Dunnett's T3 was used when homogeneity was missing. P<0.05 indicated significant. It was observed that Annexin-V/PI staining cells in each group survival and apoptosis with flow cytometry. The survival apoptosis and necrosis rate were recorded to analysis.Results:After the passage and purification of BMSCs, the cells growed adherently with pindle-shaped character, and showed cytoplasmic uniformly stained by CD29,CD44,CD90 with immunofluorescence staining, opposite to the cytoplasm no-stained by CD34. Cells nuclei showed uniform blue fluorescence with Hoechst33342 counter-staining. Of the BMSCs from the 2-6 passage, the surface marker were CD44,CD29,CD90 positive, and CD34 negtive. Moreover after different concentrations of glutamate and BMSCs were co-cultured 24h, apoptosis and necrosis cells increased and cells survival rate reduced. With the increased concentration of glutamate, there were nucleus of thick-stained, fragmentation, the growing numbers of non-uniform. The cells survival rate were obviously reduced, the apoptosis and necrosis rate was significantly increased. The cells survival rates of group A,B,C,Dwere respectively 99.11±1.5,83.56±5.6,58.76±5.9,28.29±7.9. There were statistical difference among groups (F=1371.232, P=0.000). There were statistical difference among groups (P＜0.05). The cell survival rate of all the treated groups were lower than that of the control. Moreover, among the groups of various concentration glutamate, the cell survival rates are also statistically significant difference. With the glutamate concentration increased, cell survival rate decreases. With different concentrations of glutamate and BMSCs co-cultured for 24h, it showed that for normal BMSCs, a small amount of necrosis and apoptosis of cells would be seen, normal cells was 88.4%, necrosis cells was 4%, apoptotic cells was 3.5% with Annexin-V/PI fluorescence staining by flow cytometry. In group B, there were 42.3% normal cells,40.9% necrotic cells,11.6% apoptotic cells, in group C, there were 40.8% normal cells,44% necrotic cells,6.8% apoptotic cells; in group D, there were 37.6% normal cells,47.6% necrotic cells,6.9% apoptotic cells.Conclusion1. Bone marrow stromal cells were obtained with density gradient centrifugation and adherent cultivated methods. After the passage, the cells could grow adherently, be purified in high degree and proliferate prosperitily;2. Rat bone marrow stromal cells express marker proteins CD 29,CD 44,CD90, not CD34;3. Glutamate could induce apoptosis and necrosis of BMSCs, with the increase of glutamate concentration, the cell apoptosis and necrosis rate increased.Chapter II The effect of granulocyte colony-stimulating factor on glutamate-induced apoptosis of BMSCsObjective:To obtain the apoptosis rate of BMSCs by co-cultured with different concentration G-CSF followed by glutamate.Methods:Bone marrow stromal cells were cultured in vitro for purification and amplification by application of density gradient centrifugation from the rat femurs and tibias. Groups were divided into A,B,C,D, which were respectively on behalf of normal BMSCs and co-cultured with 0,10,50ng/ml granulocyte colony-stimulating factor followed by glutamate. With the Hoechst33342 fluorescence staining, it was observed and recorded that the numbers of normal and abnormal cells from 50 cells by fluorescence staining under fluorescence microscopy through the application 0,10,50 ng/ml of G-CSF and BMSCs co-cultured for 6h, then followed by glutamate for 24h, contrast to group A. The cell survival rate were recorded and analysied with one-way ANOVA in each group. LSD was used when homogeneity of variance was assumed and Dunnett's T3 was used when homogeneity was missing. P<0.05 indicated significant. It was observed that survival, apoptosis, necrosis rate of cells by Annexin-V/PI staining cells with flow cytometry in each group. The survival apoptosis and necrosis rate were recorded to analysis.Results:There were statistical differences of the cell survival rate among groups (F= 1243.549, P=0.000), the homogeneity of variance is missing. And there were statistical differences between any groups with 30mmol/l glutamate co-cultured and group A (P＜0.05). Among the groups with glutamate, the cell survival rate of group D was higher than that of groups B and C, (P＜0.05). As well as there was no significant difference of the cell survival rate between the groups B and C (P>0.05). Under Annexin-V/PI fluorescence staining by flow cytometry, it showed that in group A, normal cells was 88.4%, necrosis cells was 4%, apoptotic cells was 3.5%; in group B, there were 40.8% normal cells,44% necrotic cells,6.8% apoptotic cells; in group C, there were 40.8% normal cells,44%necrotic cells,6.8% apoptotic cells; in group D, there were 57.4% normal cells,31.9% necrotic cells,3.9% apoptotic cells.Conclusion:G-CSF of the certain concentration have the effect of reducing the apoptosis and necrosis of BMSCs and enhance the cell survival in vitro.ChapterⅢThe combination of bone marrow stromal cells with granulocyte colony-stimulating factor for the spinal cord transection in ratsObjective:To establish the model of the spinal cord transection in rats. The neurological functional recovery was evaluated the combination effect of the transplantation of BMSCs with the treatment of granulocyte colony-stimulating factor. Methods:Bone marrow stromal cells were cultured in vitro for purification and amplification by application of density gradient centrifugation and then adherently cultured from the femurs and tibias of the SD rats. The cells were observed and marked for the transplanation under phase-contrast microscopy with cell markers Hoechst33342. Sixty female SD rats were randomly divided into four groups (each =15), including:group A (control group), the rats were transected spinal cord surgery with the injection of saline; group B (G-CSF treatment group), the rats were transected of the spinal cord injury with the subcutaneous injection of G-CSF 50ng/kg/d for the five sequent days after the operation; group C (BMSCs treatment group), the rats were transected of the spinal cord injury with the direct injection of 10 ul BMSCs composed of 3×106 cells; group D (BMSCs+G-CSF group), the rats were transected of the spinal cord injury with the direct injection of 10 ul BMSCs composed of 3×106 cells and subcutaneous injection of G-CSF 50ng/kg/d for the five sequent days after the operation. After operation, nine rats were selected for the BBB motor functional score every week for the sequent eight weeks in each group, and the data were recorded and analyzed with repeated measures analysis of variance. Morever, three rats of each group were selected for immuohistochemical staining, HE staining to observe the changes of the lesion of the spinal cord injury. The caspase-3 positive cells at 2nd week and NSE, GFAP positive cells at 4th week were recorded and analysed with one-way ANOVA. LSD test were used when homogeneity of variance was assumed and Dunnett T3 test was used when homogeneity of variance was missing, P＜0.05 indicated significant difference.Results:1. Neurological function assessment:During the experimental process,2 rats died in the control group,1 rat died3 in G-CSF group,3 rats died in BMSCs group, the data of the dead were taked away, and the same experiment rats were made complemently, according to each group respectively. All the obtained scores at every week were analysed with repeated measures analysis of variance. One-way ANOVA were used among groups on every week. The differences of the scores were found among groups (F=36.471, P=0.000), and that of the measures time were also found (F=157.670, P=0.000). There was interaction between two of them (F=23.888, P=0.000). It showed that from the 3th week after surgery, the BBB score of all the treatment groups were higher than that of group A (P＜0.05), of which the score of the group D was the highest scores. There were little differences between the score of group B and group D. Among the treatment group, the BBB score of group D was higher than that of any other groups (P＜0.05).2. Histological evaluation:At the 4th week after operation, the GFAP, NSE-positive cells are founded in each group. The number of GFAP, NSE positive cells were counted and recorded in the field of 0.05mm2 under high power microscope. It showed that there were statistical difference among the NSE positive cells of all the groups (F=198.426, P=0.000), the homogeneity of variance was assumed and the GFAP positive cells of that, the homogeneity of variance was missing. The the NSE-positive cells of all the treatment groups were higher than that of the control, there were significant difference (P＜0.05). The NSE-positive cells of group D was highest and significant statistically difference was found between group D and any other groups (P<0.05). As well as, the GFAP-positive cells of all the treatment groups were higher than group A, there were significant difference. The GFAP-positive cells of group D was highest and the significant statistically difference was found between group D and any other groups (P＜0.05). And there was no difference between the group B and C. Moreover it showed that Hoechst33342 positive cells were found in the lesion of spinal cord and some of them were NSE, GFAP and Neurofilament immunofluorescence-positive staining. At the eighth week, it showed that the obvious cavity of spinal cord injury site was found in the control group with the HE staining. The cavities of spinal cord lesion in every treatment group were obviously smaller than that of the control group, and the damage extent of the spinal cord injury of the combination were smallest.3. Apoptosis assay:It showed that the caspase-3 positive cells were found in the slice of each group by immunohistochemistry at the 2nd week. There were statistical difference among the caspase-3 positive cells of all the groups (F=69.529, P=0.000), the homogeneity of variance was assumed. There were significant statistically difference between that of all the treatment groups and the control, (P＜0.05), there were no statistical difference between that of group B and C. The caspase-3 positive cells of group D was lower than that of any other groups and a significant statistical difference were found between that of the group D and anyothers (P＜0.05).Conclusion:1. Granulocyte colony-stimulating factor have the ability of inhibiting the apoptosis of the neural tissue of the spinal cord transection injury in rats;2. The implanting BMSCs can expressed the features of neuron-like, glial-like and axonal-like cells of the features in vivo;3. Better neurological functional recovery could be found with the combination of BMSCs transplantation and G-CSF treatment.
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