Research On The Treatment Of Spinal Cord Injury By Transplanting The Olfactory Ensheathing Cells Cryopreserved At The Best Time

ObjectiveSpinal cord injury (SCI) is a common and frequently-occurring clinical disease , and the disability rate of it is very high. In recent years, the efficacy of SCI repaired by local transplantation of olfactory ensheathing cells (OECs) is very good[1], and it is considered as one of the best methods for the treatment of SCI.. The OECs of clinical transplantation to treat SCI is derived from the olfactory bulb of aborted fetuses at present, its resource is few, and the specific time and location is uncertain, the patients who wait for clinical transplantation have the same problem,so the cell sources cannot engage very well with the clinical transplantation requirements. To solve the problem, we have been researched on the relationship between the time of cryopreserved OECs and the efficacy .This experiment is based on it , treat the SCI by local transplantation with OECs which were cryopreserved for 3 months, contrast with local transplantation of fresh OECs from morphology and behavior. To investigate the efficacy of SCI repaired by local transplantation of OECs which are cryopreserved , and provide a theoretical basis for the clinical transplantation.Materials and methodsTake olfactory bulb of healthy male wistar rats about two months, cultivate and purify the OECs,collect them when they are in the logarithmic growth phase. Take the 5%DMSO-6%HES as the low temperature protectant of the OECs,conserve them in liquid nitrogen for 3 months ,then resuscitate the OECs by freezing them slowly and thawing them quickly,and mark them partial with hoechst, then transplante them into the injured site of the SCI hemisection model. The Wistar rats were divided into 4 groups randomly: fresh OECs group(A), cryopreserved OECs group (B), DF12 medium group (C) and control group(D) . The functional recovery of spinal cord injury was observed with combined behavioral score (CBS) and pathological anatomy at different phases. The tissue sections were made at 5 and 10 weeks postoperatively to observe the axon regeneration and the survival of OECs.ResultsWe can get high purity OECs by applicating the purify and culture method which combine differential adhesion and Ara-c inhibition,the cell body of the OECs is three forms, spindle-shaped, flat round and multiple ecphyma,and the immunohistochemist -ry of NGFRP75 and GFAP is positive reaction.The CBS showed the score decrease with the extension of the time in 1-10 weeks after transplantation. Group A and B change significantly,and the rats in group A and B have more improvement than that of group C and D at series of phases after 2 weeks of operation (P<0. 05), and there is no significant difference between group A and group B (P> 0.05).From the histology, we can see there are regenerated axons in every group by HE staining and silver staining. Compared with group C and group D, there are more regenerated axons in group A and group B, and the nerve fibers pass through the injury area, the difference between them is not significant.From the immunohistochemis -try, we can find the masccline staining area in the injury area of group A and group B both at 5 and 10 weeks, the OECs are yellow, and there are lots of regenerated axons around the OECs, some of them pass through the injury area, and there is no significant difference between group A and group B,we cannot find the masccline staining area in group C and group D.We can find the marked OECs in group A and group B with fluorescence microscopy at 5 weeks after operation,and there are no fluorescent particles in group C and group D; there are no obvious fluorescent particles in specimens of every group at 10 weeks after operation.Conclusion1.Taking the 5%DMSO-6%HES as the low temperature protectant of the OECs,and conserving them in liquid nitrogen is an effective method of cultivating and cryopreserving the OECs.2.The effect of treating SCI by local transplantation of OECs cryopreserved for 3 months is good,it has same effect with local transplantation of fresh OECs, the Neural function of the injuried spinal cord recover obviously,and the transplantated OECs can survive and migrate in the spinal cord area.3.The effect of treating SCI by local transplantation of OECs cryopreserved for 3 months is good, ,it solve the problem that the cell sources cannot engage with the clinical transplantation requirements, and provide academic foundation for clinic practice .