Experimental Study On Treatment Of Oldish Spinal Cord Injury With Transplantation Of Autologous Marrow Mesenchymal Stem Cells And Nerve Growth Factors

Objective: Through the establishment of New Zealand white rabbits old spinal cord injury model, and take it's autologous bone marrow mesenchymal stem cells cultured in vitro, proliferation at the same time, transplant it into spinal cord injuried and applicate of nerve growth factors at the same time at the second weekend after injuried, then observe the experimental animals body strength, feeling sphincter function at the six weekend after transplantation experiment and give score to them, and analysis the final pathology sections of spinal cord tissue image, collect the data of them for statistical analysis, and contrast the results to the conventional treatment methods for the control group, Comparative evaluation the effect of caused the functional recovery of the spinal cord of the oldish spinal cord injury in rabbits through the treatment of the autologous bone marrow mesenchymal stem cells transplantation with the nerve growth factors.Give experience for the clinic treatment of oldish spinal cord injury through the animal tests. Methods: 1.Marrow mesenchymal stem cells'harvesting and cultivation:All are extracted from the bone marrow of adult New Zealand white rabbits'autologous iliac crest, from each side of New Zealand white rabbits'autologous iliac crest respectively in order to guarantee cell concentration, and convert the direction in the process of taking suction marrow mesenchymal stem cells from bone marrow, 1 ml each side of, 2 ml total, and labeled. All received are planted in the bone marrow cell culture plates,Mediums are DMEM containing 20% fetal bovine serum solution. Incubator's temperature is 37oC, containing 5% CO2, 100% saturation humidity. Passaged cell density is 1×10 6 / cm2. Waiting theyare covered with the third-generation cell, transplantate them to spinal cord. 2. Experimental animals'grouping and handling: 36 New Zealand rabbits were randomly divided into A, B, C three groups medially, Group A for the control group, they are treated only with conventional treatment after they are resulted in spinal cord injury. group B is the autologous bone marrow mesenchymal stem cells transplantation group. Group C is the combined treatment group with autologous bone marrow mesenchymal stem cells transplantation and nerve growth factors. And observe the animal behaviours during experimental, and score them's function of spinal cord at the six weekend after transplantation. All experimental animals were killed six weeks later after transplantation, making spinal cord tissue specimens, observed by light microscopy and image analysis the tissue of spinal cord.Results: 1 Mesenchymal stem cells'growth etat: It began to cultivate a little bottle adherent cells in inverted microscope observation after primary cells were inoculated in 24h, cultured for 3~4d, they were begin to growth colony, the growth of single cells were short shuttle or polygonal shape, with the extension of time for training, adherent cells markedly increased colony is gradually increasing, a uniform shape, radiation array colony. Cells division and proliferation is very clear, extending vary in length and thickness of the many forms of inequality processes, nuclear, nuclear and ill-defined contours, nucleolus clear after 5 to 7 days, we can see that more of the split. With the cell suspension for an increase in the number of gradually being removed, about 7 to 10 days, a single cell fusion, covered the entire culture bottles. 2. Experimental animal behavior: A week later the experimental group and the control group there was no significant difference, since after two weeks, group B and group C increased movement of animals, double hindlimb muscle strength, pain and sphincter function of a certain recovery, Double-A group, the situation did not improve hind legs at six weeks, but group B and group C double experimental animals hindlimb muscle strength, pain and sphincter function recovery is better than before, the most animals urinary function independently, group C is more obvious than group B. 3. Spinal cord biopsy specimens observed: axonal atrophy necrosis or apoptosis in group A after spinal cord injury in microscope, nuclear condensation, fragmentation and dissolution, damage the local inflammatory response bleeding, formation of syringomyelia, glial scar formation. The experimental group B, group C, see a newborn neurons and glial fibrillary cells, the number of syringomyelia significantly less than that in group A, necrosis and shrinkage of neurons less than hat in group A, and Group C is good in Group B.Conclusions: 1. Centrifugal Culture and bone marrow adherent culture method is the good method to obtainmesenchymal stem cells. The bone marrow mesenchymal stem cells have goog capability to cultured in vitro and proliferation. 2. When Autologous bone marrow mesenchymal stem cells cultured in vitro and proliferated in extraorgan are transplated into the oldish spinal injury micro-environment, they can survive in the damaged areas, migration, and integration with the spinal cord, which according to the local micro-environment division, first differentiated progenitor cells into neurons and glial progenitor cells, and then differentiate into neurons and glial cells to replace damaged neurons, the transplantation of cells secreted factor to promote axonal regeneration, and promote axonal regeneration and injury and distal axon establish contact and the partial resumption of the spinal cord conduction, autologous bone marrow mesenchymal stem cells without immune rejection problems, has good therapeutic effect. 3 .Nerve growth factor can protect damaged neurons damaged neurons promote axonal sprouting regeneration, axon regeneration induced by the growth of nerve fibers, the transplantation of bone marrow mesenchymal stem cell survival and differentiation has obvious role, and Autologous bone marrow mesenchymal stem cells in the spinal cord injury of local survival, effective inhibition of glial scar formation is conducive to nerve fiber regeneration and long all, the two have a certain synergy, mesenchymal stem cell transplantation in the treatment of nerve growth factor old spinal cord injury than simply autologous bone marrow mesenchymal stem cell transplantation in the treatment of more desirable. 4. Mesenchymal stem cell transplantation could be a partial resumption of old spinal cord injury in experimental animals function, the joint application of nerve growth factor in the treatment of chronic spinal cord injury in animal experiments for the improvement of the spinal cord function of a better role to be effective in alleviating paraplegia and improve the level of spinal cord function, but the regeneration of nerve cells is still not completely injury replacement neurons restore the function of the spinal cord, spinal cord injury on the subject of old remains to be further studied.