| Background: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.Embryonic stem cells, neural stem cells and olfactory ensheathing cell transplantation is currently on a hot, but existing problems such as the lack of sources, and ethics and immune rejection, and autologous bone marrow-derived mesenchymal stem cells (BMSCs) transplantation can solve the above problems.BMSCs as a multi-potential stem cells have significant therapeutic effect to the loss of function caused by central nervous system injury or trauma. It has an incomparable advantages with the other source cells: (1) Easily acquired, can be acquired from the bone marrow of patients ; (2) Rapid increasing in vitro culture, and differentiated by inducion;(3) in the host tissue can long-term survive and integrate; (4) Autologous transplantation can avoid immune rejection reaction; (5) a genetic engineering process; (6) in a variety of ways, including intravenous injection, different parts of the central nervous system injury cell transplantation. Therefore, BMSCs is an ideal source of cells for diseases of the nervous system cells and gene therapy to provide a new way of thinking and broad prospects.Brain-derived neurotrophic factor (BDNF) as the second member of the nerve growth factor (NGF) family. BDNF has effect on dopaminergic neurons, cholinergic neurons, and a wide variety of neurons. It can promote more stem cells into neuron-like cells. Local injection of neurotrophic factor, has a certain degree of risk, and not sustainable over a longer period on high concentration. Application of systemic intravenous dose necessarily needs to significantly increase, and the specific anatomical structure of central nervous system lows the utilization of exogenous administration, which can not reach the therapeutic concentration because of the destruction of local micro-circulation. This could require a new method to maintain releasing neurotrophic factor in the long-term on a high concentration in local injury.BMSCs transfected by BDNF gene, can repeatedly passage and amplificate, and secrete a lot of production of nerve growth factor, and BMSCs induced to differentiate to nerve cells, so as to develop a new practical, possibily breakthroughing method to in the treatment of spinal cord injury by stem cell transplantation and neurotrophic factor gene therapyObjective:1, Investigate isolation, purification, culture by density gradient centrifugation and differential BMSCs adherent, in vitro. Research proliferation, differentiation, as well as immunology and other biological characteristics of BMSCs .2, Explore the the methods of constructing the plasmid total expressing BDNF and GFP and transfecting BMSCs, and the biological characteristics of BMSCs-GFP, GFP-BMSCs-BDNF.3, Investigate the Transwell co-culture methods of BMSCs, BMSCs-GFP, BMSCs-BDNF- GFP with DRG and, and the role of promoting the growth of axons of DRG.4, Evaluate BMSCs-BDNF-GFP transplantation in the treatment of rats with spinal cord injury.Methods:Partâ… 6-week-old SD rats, using Ficoll separation by density gradient centrifugation, isolate BMSCs from bone marrow. Through repeated subcultured and differential adherent to purificate the cells, the cells were high purity, biological traits stability, and the proliferation strong . And focus on the observation of the BMSCs morphological changes, cell activity, cell growth curve, surface marker of immune cells and the detection of osteogenic differentiation, and so on. Partâ…¡Mainly study obtaining BDNF sequence from the fetal rat brain tissue, constructing recombinant plasmid pEGFP-IRES2-BDNF total expressing BDNF and GFP. Lipofectamine 2000-mediated transfecting BMSCs, acquired BMSCs-BDNF-EGFP with stable and efficient GFP and BDNF expressing. Research the biological characteristics of BDNF-EGFP-BMSCs by ELISA and other methods.Partâ…¢Acquire dorsal root ganglions (DRG) from the pregnant 14d fetal rat, to establish the embryonic rat dorsal root ganglia Isolation and purification system in vitro. DRG co-cultured with BMSCs, BMSCs-EGFP, BMSCs BDNF-EGFP by 0.4 urn Transwell culture plate. Observating DRG's axons growth, Verificate cell activity by inverted microscope and immunohistochemical method.Partâ…£Animal grouping and preparation and handling of the damage model: hemisection of the rat spinal cord injury animal model, transplantation of saline, BMSCs, BMSCs-EGFP, BMSCs-BDNF-EGFP respectively, the results of experimental observations: (1) Behavioral observation: BBB score; (2) Animal perfusion, spinal cord slices. (3) Immunohistochemistry analysis; (4) BDA anterograde tracing.Statistical analysis.Results:1, Purificated BMSCs by density gradient centrifugation and differential adherent, identificated by flow cytometry instrumentation and biological markers, living cells of 95 to 100%, the purity of 99%; after induction, of alkaline phosphatase staining was positive, positive staining calcium nodules.2, successfully constructed recombinant plasmid pEGFP-IRES2-BDNF total expressing BDNF and GFP, and successfully transfected BMSCs, access to the BMSCs-BDNF-EGFP expression of BDNF increased, it is normal cells 20000 times.3, the fetal rat DRG were co-cultured through Transwell culture plate with BMSCs, BMSCs-EGFP, BMSCs-BDNF-EGFP, BMSCs, BMSCs-EGFP, BMSCs-BDNF-EGFP DRG promote the growth of axons, BMSCs-BDNF-EGFP more obvious. 4, BMSCs, BMSCs-EGFP, BMSCs-BDNF-EGFP promote the repair spinal cord injury in rats, BMSCs BDNF-EGFP-effects more visible.Conclusion1, density gradient centrifugation and differential adhesion method is a good method of separation and purification BMSCs.2, BDNF pEGFP-IRES2-GFP and the BDNF gene in a vector with a promoter coordinated high expression.3, BMSCs-BDNF-EGFP DRG axons could significantly promote growth.4, BMSCs-BDNF-EGFP can significantly promote the restoration of spinal cord injury in rats, and is becoming a ideal source of stem cell transplantation and becoming a ideal cell vector of neurotrophic factor gene therapy in the treatment of spinal cord injury.
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