|
Objective: To establish rabbits old model of spinal cord injury, and also extracts its autologous bone marrow mesenchymal stem cells cultured in vitro, proliferation, and then transplanted at 2 weeks after injury in the spinal cord injury site and also combined with nerve growth factor treatment, spinal cord functional recovery, and 6 weeks after transplantation in experimental animals limb muscle strength, sensation, sphincter functional recovery score, and finally spinal cord tissue biopsy image analysis, statistical analysis of the data, and only conventional treatment control group were compared to evaluate autologous bone marrow mesenchymal stem cell transplantation combined with nerve growth factor to promote the cause of old spinal cord injury rabbits functional recovery aspects of treatment effect. By the animal experiment for clinical treatment of chronic spinal cord injury accumulate experience. Method: 1. Mesenchymal stem cells to obtain and culture: all are from the bone marrow of adult New Zealand white rabbits with autologous iliac crest extraction, to ensure the cell concentration, respectively, from the right and left sides of each rabbits iliac crest bone marrow , and in the process transform bone marrow puncture needle direction, each side extraction 1ml, a total of 2ml, labeled number. Received bone marrow cells grown in culture plates. Culture medium containing 20% ??FBS DMEM solution. Incubator temperature 37oC, containing 5% CO2, 100% saturated humidity. Passaged cell density of 1 × 106/cm2. Subject to the third generation of cells covered conducted after spinal cord transplantation. (2) Animal grouping and treatment: 36 New Zealand white rabbits were randomly divided into A, B, C3 group, A group 12 for the control group, resulting in spinal cord injury model using only conventional treatment after; B group 12 to autologous bone marrow Mesenchymal stem cell transplantation group. Group C, 12 autologous bone marrow mesenchymal stem cell transplantation combined neurotrophic factor treatment groups. Each period were observed behavior of laboratory animals, and 6 weeks after transplantation for spinal cord function score was measured. 6 weeks after transplantation all animals were sacrificed, the production of spinal cord tissue slice specimens for light microscopy and image analysis organization. Result: a mesenchymal stem cell growth: primary cells inoculated 24h, inverted microscope, cultured bottom began to appear a little adherent cells were cultured 3 ~ 4d, visible cells begin to show colony growth, a single growing cells were short shuttle shaped or polygonal, with the incubation time, adherent cells increased gradually increasing colony, there were highly homogeneous, arranged radially colony. Cultured for 5 to 7 days after cell division and proliferation is very clear, extended lengths, uneven thickness of the variety of forms of projection, large nucleus, nuclear contour is unclear, clear nucleolus, showing more split phase. With the suspension cell medium was changed gradually increased the number to be removed, about 7 to 10 days, the cells fused into a single layer, covered the entire culture flasks. (2) the performance of animal behavior: a week after the experimental group and the control group had no significant difference, since the beginning after two weeks, B, C group animals begin to exercise increased muscle hind limbs, pain and sphincter function has a certain recovery, A group of animals at 6 weeks, no significant improvement status hind limbs, while B, C group of experimental animals hind limbs muscle strength, pain and sphincter function significantly compared with the previous recovery, most of the animals have spontaneous voiding function, C group is more than B group is obvious. 3 slices of spinal cord specimens were observed: in group A microscope axonal atrophy after spinal cord injury necrosis or apoptosis, nuclear condensation, fragmentation and dissolution of local inflammatory injury bleeding reaction to form syringomyelia, glial scar. While the experimental group B, C group saw a newborn neurons and glial fibrillary cells, syringomyelia number significantly less than group A, necrosis and atrophy of neurons also less, and group C was better than the B group. Conclusion: a bone marrow cultures were centrifuged and adherent culture method are combined is to obtain bone marrow mesenchymal stem cells in a good way, bone marrow mesenchymal stem cells with good vitro proliferative properties. 2 In vitro proliferation after autologous bone marrow mesenchymal stem cells into the spinal cord microenvironment old injury, it can survive at the site of injury, migration, and integration with the spinal cord, according to which the local microenvironment differentiation occurs, first progenitor cells differentiate into neurons and glial progenitor cells, and then differentiate into neurons and glial cells, replace damaged neurons transplanted cells secrete factors to promote axonal regeneration and promote axonal regeneration and with the injury distal axons to establish contact, the partial restoration of spinal cord conduction function, autologous bone marrow mesenchymal stem cells without immune rejection problem, has a good therapeutic effect. 3 of nerve growth factor can protect damaged neurons, promotion of damaged axon sprouting regeneration of nerve fibers to induce regenerative axon growth of transplanted bone marrow mesenchymal stem cell survival and differentiation of a significant role in promoting and Autologous bone marrow mesenchymal stem cells can be damaged spinal cord partial survival, effectively inhibit glial scar formation, is conducive to nerve fiber regeneration and ingrowth, both have a certain synergy, bone marrow mesenchymal stem cell transplantation combined with nerve growth factor treatment old spinal cord injury than simply autologous bone marrow mesenchymal stem cell transplantation treatment effect is more ideal. 4 bone marrow mesenchymal stem cell transplantation can result in partial restoration of spinal cord injury in experimental animals with old features, combined with nerve growth factor treatment of chronic spinal cord injury in experimental animals for the improvement of spinal cord function have a more desirable effect, can effectively reduce the The extent and improve paraplegic spinal cord function, but the regeneration of nerve cells is still not completely replace damaged neurons to restore the function of the spinal cord, spinal cord injury on the subject of old remains to be further studied.
|