| [Objective]The Schwann cells acquired and purified from Wistar rats and the human cord blood mononuclear cells obtained by means of Ficoll method were co-grafted into the lesion site of injured spinal cord in order to discuss the effect of cells co-grafting for the treatment of spinal cord injury and evaluate its efficacy.[Method]The bilateral saphenous nerves were cut out from the Wristar rats. Dual-enzyme digestion method combined with mechanical separation were used for the culture of SC in vitro;The SCs were purified by means of low concentration of collagenase,rapid trypsin digestion together with differential adhesion.After that, the growth characteristic of SCs was observed and then the identification of SCs was carried out in vitro.Forty Wristar rats were made into spinal cord injury model at T10 by MASCIS Impactor ModelⅡand then assigned into 4 groups randomly and evenly, which were DMEM control group,HCMNCs graft group,SCs graft group, HCMNCs and SCs co-graft group.After one week of successful modeling,DMEM, cultured SCs,cultured HCMNCs and SCs-HCMNCs were transplanted into the lesion site of injured spinal cord respectively according to the grouping.BBB score was used to evaluate the hindlimb recovery from 1 week after modeling to the 12th week.At 12th week after modeling,footprint analysis was used to assay the interlimb coordination and angle of rotation of hindlimb.After 12th week of surgery,3 rats in each group were chosen for the 10%BDA nerve anterograde tracing,two weeks after labeling,the rats were sacrificed.Their injured spinal cord was taken out to carry out the 5μm fast frozen section.Then,BDA developing,immunohistochemistry stain and HE stain were carried out.The BBB score in four groups was analysied by Repeated measures analysis of variance.The digital image of immunohistochemistry stain was processed by the software of Image pro plus 5.0 for the comprehensive evaluation of the injured spinal cord.[result]SCs pass over four generations after isolation and purification in vitro.The cells were bright,pointed at both ends,expanded in the middle of a long spindle, which showed a well growth state.After being stained with S-100,the cells showed green colour in their cytoplasm and the shape of bipolar or multipolar spindle morphology,nuclei were bright,their processes were dark.Within three weeks after injury,no statistic difference existed among four experimental groups in the evaluation ofhindlimb functional recovery until the 4th week after injury(p＜0.01), the BBB score is the highest in cell co-graft group.BBB score in SCs graft group and HCMNCs graft group is higher than that in control group,but lower than that in cell co-graft group.In footprint analysis,statistic difference existed among four groups in the evaluation of interlimb coordination and angle of rotation(p＜0.01),the improvement is the best in cell co-graft group.The result of histological observation is in accordance with functional test.HE and BDA stains showed the injured cavity is obviously smaller in cell co-graft group than that in other three groups,and more regenerated nerve fibers pass through the injured cavity.NF-200 stain demonstrated that the positive response area is the largest in ceil co-graft group,its IOD value is the highest(P＜0.01)。[conclusion]SCs secrete numerous neurotrophic factors,promote the demyelinated nerve fibers for remyelination,and final promote the nerve regeneration and functional recovery after spinal cord injury;HCMNCs can differentiate into neurons, astrocytes,and oligodendrocytes after being grafted into the injured lesion,filled in the injured cavity and take benefit for the axons extend and synaptic connection. After cell co-graft,SCs can promote more HCMNCs differentiate into neurons to fill in the injured cavity,which evidently improve the functional recovery after spinal cord injury.
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