| Spinal cord injury(SCI) is a common injury in transport, mining accidents, sports injuries and warfare nowadays. Most of the injured are young people. The paraplegia caused by SCI not only seriously affects the physical and mental health of patients, but also put up to families and the community tremendous economic, human and spiritual burden. For a long time the repair of spinal cord injury has been plaguing human health problems, and the world scientists are working on the focus, but still lack of practical and effective therapy methods. Curing spinal cord injury is always a difficult and hot point in neuroscience research. It's believed that normal mature mammalian central nervons system couldn't regenerate after injured, because of poor regeneration capability of neuron and the inhibitor form glial microenvironment. Since 1981 the neuroscientist confirmed that the repair of the central nervous system structure after injury was possible, the study on repair of SCI has been becoming hot spot in many academic institute. The reasons that interfering axon regeneration after SCI were extremely complicated. First, primary and secondary injury on spinal cord caused a great of spinal cord functional neurons apoptosis and necrosis, which making the regeneration of axons difficulty. Second, a great quantity secretion and release of myelin-associated inhibitor and axon growth inhibition protein after SCI. The secondary glial scar impeded the axons growth and properly connected. Third, the decreased secretion of many neurotrophic factors resulting from partial cells apoptosis destroyed the micro-environment benefiting for neurons repair and axon regeneration. The measures of repairing SCI currently are mainly the following aspects:drug treatment, cell transplantation, tissue transplantation, improve the local micro-environment and union treatment. The traditional high-dose corticosteroids(methylprednisolone) impacting treatment could effectively reduce the secondary inflammation after SCI, and protect the residual damaging neurons and prevent the further apoptosis. At present, the studies claimed that lithium chloride(Licl)and the immunosuppressant FK506, and so on, could protect neurons from injury and could effectively promote axon regeneration. Cell transplantation is currently a hot research, the principle is to use some seed cells secreting a variety of neurotrophic, adhesion agents, nutrite and protect the neuron after injury, and then induced axon regeneration and re-myelinization to repair SCI. Previous studies have provided a more mature seeds cells for SCI, such as: embryonic stem cells(ESCs), schwann cells(SCs), olfactory ensheathing cells (OECs), neural stem cells NSCs), bone marrow mesenchymal stem cells(BMSCs), the umbilical cord blood stem cells(UCBCs). A large number of studies have confirmed that cell transplantation facilitates the restoration of spinal cord function. With the tissue engineering rapid development, a more advanced concept of SCI repairment appeared gradually that repair of SCI must include the organization levels reconstruction. Integrating effective treatments of SCI repair strategy, we could reduce glial scar formation at the same time induce the directional axon regeneration. At present, there were two engineering scaffolds applicating in spinal cord tissue, one was biodegradable polymer synthetic biological material, such as polylactic acid, and polyglycolic acid copolymer, another was autologous or allogeneic tissue transplantation such as allogeneic embryonic spinal cord tissue, autologous muscle fiber frame, free of peripheral nerve(FPN), or the free vascularized peripheral nerve(VPN) tissue transplantation to repair spinal cord injury, and achieved a certain degree of effect. Another key factor for axon regenerate obstacle after SCI is the appropriate axonal regeneration micro-environment has been damaged, and myelin-derived protein(myelin,MAG, Nogo-A, Omgp) which inhibited the axon regeneration were greatly secreted, and lacking of various neurotrophic factor (BDNF and GDNF, NGF, NT3)and the glial scar formation hindered axon regeneration. To this end, a new therapy strategy Combined with several methods in different mechanism was used, which may have a better synergy to promote nerve repair after SCI. Accordingly,this study was designed to apply lithium chloride combined with transplantation of human umbilical cord blood mesenchymal stem cells and acellular spinal cord scaffold to repair SCI. From this study, the nerve regeneration and function recovery of SCI animal will be investigated, at the same time, the possible mechanism of recovery in SCI will be discussed.Obsjective:1. To investigate the method and conditions of isolation, proliferation of multipotent mesenchymal stem cells(MSCs)from human umbilical cord blood in vitro, and he possibility of inducing human umbilical cord blood mesenchymal stem cells (MSCs) to differentiate into neuron-like cells.2. To investigate the effects and possible mechanisms of treating complete transected spinal cord in rats through lithium chloride combined with transplantation of human umbilical cord blood mesenchymal stem cells and acellular spinal cord scaffold3. To explore a method for fabricating the acellular spinal cord scaffold and to observe the construction features of the scaffold.4. To investigate the possibility of inducing human umbilical cord blood mesenchymal stem cells (MSCs) to differentiate into neuron-like cells by lithium chloride (LiCl) in vitro.Methods:1. Human umbilical cord blood was collected from mature neonates. All samples were obtained sterilely with 20 U/ml heparin. The cord mononuclear cells were isolated with lymphocyte separation medium (density 1.077g/ml), then purified by wall sticking screening and expanded with slight sugar DMEM containing 15%FBS. Immunophenotypes of the cells surface were analyzed by flow eytometry. Expanded MSCs were induced to differentiate into neuron-like cells in medium added with basic fibroblast growth factor(bFGF), and immonohistochemical staining was used to identify the specific protein of neuron:microtubule associated protein-2(MAP-2)。2. The third passage of the expanded MSCs were pre-inducted with DMEM containing 15%FBS and 20ng/ml bFGF for 24 hours, then induced with DMEM without serum but 3mol/L Licl for 6 days in group A. The MSCs were induced with DMEM containing 3mol/L Licl for 7 days in group B. The MSCs were normally cultured with DMEM containing 15%FBS in group C. The morphological changes of the cells were observed under phase contrast microscope. The neuron specific markers containing neuron specific enolase(NSE), microtubule associated protein-2(MAP2) and glial fibrillary acid protein(GFAP) were evaluated by indirect immunocyto-chemistry staining.3. Several Segments of spinal cord were obtained from Adult female Sprague-Dawley rats. Under operative microscopy, the fat and a part of dural matter were cutted before the extraction procedure. Segments of spinal cord obtained from rats were divided randomly into three groups: group A(frequency of vibration=80r/min), group B(frequency of vibration=120r/min) and group C (frequency of vibration=160r/min). The spinal cord Was delt with solution of Triton X-100 and with solution of sodium deoxycholate at room temperature. Then washed with distilled-water, delt with HE staining and observed under light microscope. Scanning electron microscope was used to observe the ultramicrostructure. The fabricated acellular spinal cord scaffolds were stored in 0.01mol/L PBS(PH7.4).4. At first, to establish the entire transected spinal cord injury model at T9 level in rats. Then 120 Sprague-Dawley (SD) female rats were divided randomly into six groups,20 in each group. Group A(spinal cord entire transaction), group B (spinal cord entire transaction+intraperitoneal injection of 85mg/kg lithium chloride every day), group C (spinal cord entire transaction+hUCB-SCs transplantation), group D (spinal cord entire transaction+hUCB-SCs transplantation+intraperitoneal injection lithium chloride), group E (spinal cord entire transaction+acellular spinal cord scaffold and hUCB-SCs transplantation), group F (spinal cord entire transaction+ acellular spinal cord scaffold and hUCB-SCs transplantation+intraperitoneal injection lithium chloride). At 1 day,3day and the end day of every week post-operation, a behavioral testing was performed weekly upon each hindlimb of all animals according to the BBB scoring system. At the 8th week, all animals were sacrificed and the spinal cords were taken out for morphological observation. Tissues in SCI sites were observed with H&E staining and Brdu nuclear labeling to identify the survival and migration of SCs. With immunocyto-chemistry staining to identify the differentiation of neuron-like cells and expression of the neurofibras. With fluorescent-gold(FG) spinal cord retrograde tracing to observe the regeneration and distribution of spinal nerve fiber. The treatment effects of spinal cord injury were identified comprehensively.Results:1. The hUCB-SCs could be isolated successfully from the UCB with density gradient centrifugation. Flow cytometry analysis showed that the cells were positive for CD90 (neural cell antigen)and CD29, CD105 (MSC-specific surface markers), while negative for CD34 (hematopoietic stem cell antigen)and CD45 (leukocyte common antigen). MSCs differentiated into neuron-like cells induced for 14 days by 20ng/ml bFGF and the differentiated cells faintly expressed MAP-2.2. After inducted for 3 days, morphological changes were observed obviously in group A and B.6 days later, the differentiated cells showed typical neuronal morphology. The expression of NSE and MAP2 were positive for the majority cells in group A and B, but that of group A [(73.6±7.8)%, (75.5±8.5)%]were obviously higher than group B [(31.0±4.3)%, (3.5±5.0)%]. few expressed GFAP in both groups.3. Acellular spinal cord scaffold can be fabricated by chemical extraction. The three-dimensional(3D)structure of the acellular spinal cord scaffold are kept intact, in which there are bundles of extracellular matrix (ECM) fiber which aligned lengthways were interlaced with transversal matrix fibers.4. It could be observed that Brdu marked hUCB-SCs survivaed and migrated in the spinal cord postoperative 8th week in groupC, D, E and F. The survivaed hUCB-SCs in group D and F were extremely more than that in group C and E. Through FITC fluorescence labeling NF-200 positive nerve fibers, No NF-200 positive nerve fibers could be observed in group Aand B; NF-200 positive nerve fibers in group D and F were extremely more than that in group C and E. Some continuited nerve fibers through injury district could be observed in group D and F. Fluorogold(FG)retrograde tracing show that a small amount of pyramidal cells were labeled by FG in group E and F. The nerve pyramidalcells marked by FG occasionally were observed in group C and D, the remaining two groups were not apperence. There were significant differences of BBB Score of hindlimb functional movement among all groups except group Aand B. The BBB Score of hindlimb functional movement were better in group F than that in group E, better in group D than that in group C.Conclusions:1. The results suggest that MSCs can be obtained from HUCB. MSCs from HUCB can be induced to differentiate into neuron-like cells and faintly express MAP-2 in vitro.2. Licl could induce the human umbilical cord blood MSCs to differentiate into neuron-like cells in vitro. Licl combined with bFGF could improve the induced effects.3. Acellular spinal cord scaffold can be fabricated by chemical extraction. The native 3D structure of the ECM in the scaffold maybe provide the structural foundation for inducing effectively the neurons and axons to directionally grow and migrate.4. Licl could improve survival and differentiation into neural cells of the human umbilical cord blood in injury district. Acellular spinal cord scaffold could bridge the both stumps of the injured spinal cord by means of "alete butt joint", stop the in-migrating of the peripheral tissues around the spinal cord, guide the directional growth and migration of the neural cells and axons. Licl combined with transplantation of human umbilical cord blood mesenchymal stem cells and acellular spinal cord scaffold could improve the functional recovery of the hindlimbs in the complete transected spinal cord rats.
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