| Objective:The study aims to establish the steady and easily replicated models of spinal cord injury in SD rats, and to investigate the effect of bone marrow mesenchymal stem cells (BMSCs) transplantation combined with erythropoietin (EPO) for repairing spinal cord injury.Methods:1.Femurs and tibias were aseptically removed and bone marrow flushed from each piece. Bone marrow was placed in100mm culture dish in Dulbecco’s Modified Eagle Medium supplemented with10%of foetal bovine serum. The culture medium was changed once per day for three days in order to remove non-adherent cells. Then the culture medium was changed twice a week and cells were passaged when they reached confluence. Cells in passage2and passage3were characterized by their specific surface antigen expression with immunocytochemistry against CD29(+), CD90(+), CD34(-) and CD45(-).2.Models of spinal cord injury were established by Hatteras Instruments PCI3000Precision Cortical Impactor.3.120SD rats were randomly divided into four groups:control group(group A,n=30), EPO group(group B,n=30), BMSCs group(group C,n=30), BMSCs and EPO associated group(group D,n=30),10ul suspension of BMSCs (5×107/ml) were given by local injection to the spinal cord in group C and group D after spinal cord injury, and EPO (5000IU/Kg/BW) were peritoneal injected in group B and group C30minutes after spinal cord injury. Six rats in each group were scored by the Basso-Beattie-Bresnahan(BBB) locomotor rating scale to evaluate motor function on the1th、3th、7th、14th、28th day after spinal cord injury, then rats were killed to observe cell apoptosis and the expression of NF-200, what’s more, BrdU-positive BMSCs were examined in group C and group D. The data was analyzed by SPSS19.0.Results:1.These cell populations are comprised of morphologically homogenous fibroblast-like cells and present plastic-adherent property. Phenotypes of P2generation were detailed as:CD29:91.5%, CD90:96.2%, CD34:1.3%, CD45:8.0%. Phenotypes of P3generation were detailed as: CD29:98.2%, CD90:98.1%, CD34:0.6%, CD45:2.0%.2. The cell growth curve indicated cell proliferation reached maximal rate at inoculation concentration of8×104/ml.3.The BBB scores were descendingly ordered as group D, B, C and A at7th、14th、28th day after spinal cord injury (P＜0.05).4. The apoptotic cells index began to rise with24hours and reached its climaxuntil3rd day after spinal cord injury before declining,Groups comparison of AI in each time was for A>B>C>D (P<0.05).5.NF-200expression decreased sharply after spinal cord injury and rebounded to rise3days later.Groups comparison of IOD in each time was for A<B<C<D (P<0.05).6. The BrdU-positive rate of group D was higher than that of group C (P<0.05).Conclusions:1.Bone marrow mesenchymal stem cells of passage3could be pertained as seed cells for spinal cord injury.2.Hatteras Instruments PCI3000Precision Cortical Impactor contributes to establishing steady models of spinal cord injury with SD rats.3.BMSCs cooperates with EPO could improve protection and restoration of spinal cord injury by reducing apoptosis, accelerating neuro-protein expression. |
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