| Spinal cord injury(SCI)is one of the most serious surgical trauma. With the rapid development of economy, traffic accidents, construction height of fall and sports accidents caused by spinal cord injury showed a rising trend year by year. Paraplegia is the most serious consequence of SCI, which gives the family a tremendous social and economic losses and burdens. Therefore, the treatment of SCI is still the focus of attention of one of the difficulties in our society and modern medical research. In addition to primary traumatic spinal cord injury directly caused by spinal cord injury, the release of harmful factors and micro-changes in the environment is also indirectly involved in spinal cord injury. Lack of the factor who promote nerve repair, release of the factor who inhibit nerve repair, glial scar formation and the persistence of such a situation cavity, allowing repair of spinal cord becomes very difficult.In particular, neuronal apoptosis after SCI, can cause the spinal cord irreparable damage. Therefore, the mechanism of cell therapy and treatment of SCI research in this field is particularly important. Stem cell transplantation is one of the treatments of SCI in recent years. Taking into account cell-derived, immune response, Ethics, and many other factors, bone marrow-derived mesenchymal stem cells (MSCs) have been more used in cell transplantation treatment.This research project includes:(1) According to adherent growth characteristics of MSCs, isolation and purification MSCs in vitro, microscopic observation of morphological changes, assay their growth characteristics by MTT, detect cell life cycle by using flow cytometry, identify the separated MSCs by detecting cell surface markers CD34 and CD44,and directed inducing differentiation of MSCs.(2) Preparation of spinal cord injury model in rats with the falling weight method which used Allen's strike bench and the Rats in each group were taken to do routine HE staining in spinal cord tissues.(3) We injected the cultured MSCs in vivo rat model of SCI via the tail vein, record scores of the rats by using modified Tarlov scoring criteria and made statistical analysis.(4) Rats in each group were randomly taken to do routine HE staining in spinal cord tissues.(5) Use the conditioned medium which simulated in vivo micro-environment cultured MSCs in order to make MSCs transform to nerve-like cell.(6) Using immunohistochemical staining and RT-PCR methods detected the expression of neuron-specific enolase (NSE), neurofilament (NF) and glial fibrillary acidic protein (GFAP) in transforming cells.(7) observed MSCs that marked by diamidino-phenyl-indole (DAPI)migrate to the spinal cord injury cases by confocal laser scanning microscope.(8) MSCs with BrDU labeled in vitro were transplanted by the SCI rats tail vein, then immunohistochemical stained the spinal cord tissue after 21 days, laser scanning confocal microscope BrDU labeled MSCs were observed the expression of neuron-specific enolase (NSE) in MSCs labeled by BrDU with laser scanning confocal microscope, in order to investigate the mechanism of MSCs treatment of SCI.The results of the experiment showed that:(1) Vein graft MSCs in rat spinal cord injury model has a definite therapeutic effect.We transplanted MSCs via the tail vein to the spinal cord injury rats, recorded scores of rats in each group after injection Id,14d,28d respectively. The conclusion is that:After treatment the score of normal group and sham group is normal. The score of the remaining three groups are improved in the 28th day after treatment. With statistical analysis, on the 7th day after treatment, scores of the model group and the placebo group had a significant deviation compared to the treatment group(P<0.01); on the 14th day after treatment, scores of the treatment group were still higher than the model group and the placebo group(P<0.05); on the 28th day after treatment, scores of the treatment group were significantly higher than the model group and the placebo group(P<0.01),but lower than the normal group and the sham operation group(P<0.001). Of course, this treatment improved motor function is not already complete. The score of treatment group compared with the normal control group still has differences, which suggest that functional reconstruction after spinal cord injury is still limited. Microscopic tissue section observed in normal control group of spinal cord gray matter and white matter structural integrity of the organization, nerve cells in the gray matter in the uniform distribution of normal morphology. In the 1st day, the injured spinal cord in a visible hemorrhage, cell swelling, cell degeneration and structure of the lack of clarity in pathological changes. Treatment group, MSCs were vein transplanted via the tail, can be seen that tissue edema reduce, inflammatory cell infiltration reduced, the nerve cells and structural are tended to re-ordered on the 14th days. Experimental results show that MSCs can improve vein graft SCI rats motor score, promote functional recovery of spinal cord of rats with spinal cord injury model has a definite therapeutic effect.(2) It was illustrated that spinal cord grinding liquid were the same as cytokine, which could induce MSCs into neurocyte directionally.Cytokines (bFGFand EGF) and spinal cord grinding liquid were successfully used to induce MSCs into neurocytes directionally in the experiment. The transformant, using histochemical stain, were express positively about NSE,NF and GFAP under laser confocal microscopy; the production of RT-PCR by electrophoresis demonstrate that, the expression of NSE,NF and GFAP of the cell which had been introduced had increased obviously compared to nulli-introduced control group. It was illustrated that spinal cord grinding liquid were the same as cytokine, which could induce MSCs into neurocyte directionally, as well that the differentiated production contained both nerve cells and neurogliocyte. In other words, MSCs traceable mesoblastema could transform into nerve cells of ectoderm, which expressed both neuronal specificity proteinum and neurogliocyte specificity proteinum.(3)the mechanism of treatment of SCI by MSCs is probably that the intravenous transplanted MSCs could be relocated to the spinal cord injury and can differentiate into neural-like cells.MSCs labeled by DAPI in vitro were injected into the model rats of spinal cord injury via the tail vein, then it can be observed by confocal laser microscopy that there are fluorescence labeled MSCs in the vascular of spinal cord injuried tissue at the 24 hour post-injection. The 5th day post-injection, the dispersion of labeled MSCs can be detected around the blood vessel and surrounding tissues injuried. The 10th day post-injection, the the wide range of dispersion of labeled MSCs can be detected around the surrounding tissues injuried. At the first 24 hour, the first 5 day and the 10 day post-injection, there are no DAPI labeled MSCs in the normal rats. This indicates that the MSCs vein transplanted can be directed to the spinal cord injury in the migration and settlement.Then the green expression of BrDU and the red expression of NSE are both detected by the laser confocal microscopy, while the non-transplantation group and normal control group detected only NSE, no BrDU. This indicates that the transplanted MSCs in the injured spinal cord within the organization may apply to the neuron-like cells differentiation.This study proved that intravenous transplanted MSCs has a definite therapeutic effect on the model rat of spinal cord injury. MSCs can differentiate into nerve cells under specific conditions both in vitro and in vivo. Intravenous transplanted MSCs can be directed to the injured spinal cord at migration, and can be transformed into neuron-like cells, filling defects, which play a role in spinal cord injury. This study enriches the experimental treatment of SCI, and provide a theoretical basis and supporting data to clinical application of MSCs.The innovation points of this study:1 This experiment confirmed that intravenous transplantation of allogeneic MSCs can be used for the treatment of SCI rats, and this method can improve motor function in SCI rats, and it has a definite therapeutic effect to SCI model rat.2 In vitro experiments, it can be confirmed that MSCs can cross-mesoderm differentiate into neuron-like cells, and can express neuron-specific markers under certain conditions.3 In vivo experiments, it can be confirmed that intravenous transplanted MSCs can be directed to the injured spinal cord at migration, and filled tissue defect, and can be transformed into neuron-like cells, express neuron-specific enolase.Red and green double staining of cell transformation can be observed by laser scanning confocal microscope. It revealed the mechanism partly that MSCs could promote the recovery of SCI.
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