Studies On The Repair Of Spinal Cord Injury By Transplantation Of Olfactory Ensheating Cells

Spinal cord injury (SCI) is a frequently encountered clinical problem. It can easily lead to crippledom and seriously damages to people's health,especially serious spinal cord injury.It's treatment is still a century problem.Recent studies have proved that olfactory ensheating cells (OEC_S) can promote the regeneration of spinal cord axons by excreting several factors and forming myelin sheathes for regenerating axons.It is believed that the transplantation of olfactory ensheathing cells would be effective option for the treatment of SCI.Objcctive:To explore a effective method for OEC_S culture and purification and observe OEC_S survival and repair in vivo for spinal cord transection after the OEC_S transplantation.Methods:1. Cell culture: The primary OEC_S was cultivated with the olfactory bulb of Sprague-Dawley rats, and then the purification was done by the differing rates of attachment of the various harvested cell types and anti-Thy1.1-mediated complement lysis and free serum medium. The morphological changes of the cultured OB-OECs were observed under a phase contrast microscope at different culture time.As a result, the majority of neuron and other mixed cells were eliminated. Because only OEC_S express both GFAP and P⁷⁵ in the CNS, we can identify it by this characteristic. The purity of the OB-OECs was evaluated according to the percentage of NGFRp75 immunostaining cells.2. Animal experiment: The T₉ spinal cords of 4 months Sprague-Dawley rats was exposed, then complete transection was performed in the middle of the area in Group A and Group B. In group A (20 rats) ,OEC_S was transplanted. In group B (20 rats), DMEM were injected. In Group C (10 rats), T₉ laminectomies were done without cord transection. The functional recovery of the spinal cord injury was observed with BBB locomotion score in each double weeks. The tissue sections were done 3 months postoperatively. HE staining, silver staining ,NF and P⁷⁵ immunohistochemical staining were performed respectively to observe the pathological changes and axon regeneration. The survival of OEC_S was observed by P⁷⁵ staining under the microscope.Results:1. The differing rates of attachment of the various harvested cell types with anti-Thy1.1-mediated complement lysis and free serum medium was a efficient method of OB-OECs puritification and culture.2. The experiment showed that locomotion behaviour had improved in 4 weeks postoperatively. The BBB locomotion score of group A was higher than that of group B in all periods, statistically significant difference in 12 week after operation.3. Nerve fiber innervated injury area in the group A and B.In the cephal lesion, the number of nerve fibers of group A was more than that of group B(P<0.001), but less than that of group C(P<0.001).4. The OEC_S by P⁷⁵ immunohistochemical staining could be observed around spinal injury area through the microscope. OEC_S could migrate to 1.0cm far from the injury area.5. There was positive correlation between the number of regeneration axons and the recovery of locomotion function(r=0.981).Conclusion:1. The cell culture of the differing rates of attachment of the various harvested cell types with anti-Thy1.1-mediated complement lysis and free serum medium can obtain high purity OEC_S. Furthermore, the method is simple and economical.2. After transplantation, OEC_S can survive for at least 3 months in vivo, and can migrate from the injection sites.3. For spinal cord transection, OEC_S can promote the axons regeneration and the recovery of locomotion function in our study.