Effects And Mechanism Of Transplantation Of BDNF Genes Modified Bone Marrow Stromal Cells On Spinal Cord Injury

IntroductionBone Marrow Stromal Cells(BMSCs) are stem cells derived from mesoderm and have become the ideal gene target cells for their advantages of multiple differentiations and amplification abilities. Brain derived neurotroph factor(BDNF) is an important member in the family of neurotrophic factors(NTFs) and it can promote the survival, differentiation and outgrowth of motor neurons. In this study, the recombinant PcDNA3.1-BDNF plasmid was constructed and transduced into BMSCs by lipofectin. We observed the expression of BDNF and its effect on the differentiations of BMSCs. The rat semi-division of the spinal cord model was successfully built, and BDNF-engineered BMSCs were transplanted into the injuried spinal cord. The motor functional recovery was evaluated. Moreover, the spinal cord were detected the Caspase-3 with immunochemistry technique. We try to find the protocol of gene therapy for spinal cord injury(SCI).Methods1,Culture of BMSCs(1)Primary culture was used to culture BMSCs;(2)Immunoflurescence staining were adopted to identify BMSCs(CD44 staining);(3)Neurogenic differentiation of BMSCs, the NSE and GFAP staining were used to identify. 2,Construction of eukaryotic expression vector and obtain cell clones expressing BDNF gene stably.(1)The DNA was extracted from plasma of Norwegian rats, the eukaryotic expression vector PcDNA3.1-BDNF was constructed and identified;(2)The recombinant plasmid was transfected into BMSCs by lipofectamin method and positive cell clones were screened with G418;(3)Overexpression of BDNF gene in the transfected cells was confirmed with Western Blot and RT-PCR.3,Animal model and transplantation of cells(1)SCI model was built;(2)Rats were divided into 3 groups at random: Group A consisted of the rats without treatment; group B consisted of the rats treated with transplantation of BMSCs without genetic modification; the rats in group C were treated with the transplantation of BDNF genes modified BMSCs. Every group divided into 1day, 3 days, 7 days and 21 days phases, there are 6 rats in every phase;(3)BDNF genes modified BMSCs were transplanted into the injuried spinal cord.4,Results test(1)Neurological functional test;(2)Caspase-3 was detected with immunochemistry technique;(3)Data analysis with SPSS software.Results1,The growth and identification of BMSCs24 hours after primary culture, the cells took a round shining shape, and were adherent to the bottom dispursely. As time pass, cells were cluster-like clones appears, and maldistribution. The cells are dense and fibroblasts-like one week later. BMSCS were passaged after 80% of bottom were covered, after 2-3 passages, BMSCs were identified with CD44 with immunofluorescence, a light green fluorescence was visible under the microscope. BMSCs were observed in the same field with both bright mode and fluorescence mode, over 95% cells were CD44 positive. 2,The induction of BMSCs in vitro and immunocytochemical test24 hours after induced by spinal cord fluid and BDNF, the morphous of some BMSCs were obviously different. They became small and contracted and exhibited spherical. The postinduction BMSCs were extremely similar to the neural cells. The cells were stained immunocytochemically with NSE and GFAP antibodies after induction 14 days. Most cells were positive in NSE and GFAP and were brown. The positive rate of NSE was 79.7%±2.6%; the positive rate of GFAP is 66.8%±5.7%.The cells of control group expressing NSE, GFAP was very few.3,By using the primers we designed, the PCR product was in accordance with anticipate one. There was no DNA band amplified in the product of negative control group. Results Enzyme digestion analysis and sequencing showed that the target gene was cloned into recombinant vector.4,Overexpression of BDNF gene in the transfected BMSCs cells was confirmed with RT-PCR and Western Blot.5,Evaluation of ethology1 day after operation, all the rats in group A, B and C showed the paralysis of both back legs, and had no significant difference. 7 days after operation, the motor function of rats in group B and C are better than that in group A(P<0.05), 21 days after, the rats in group B and C can walk.6,The immunocytochemical result of caspase-31 day after operation, the positive rates of caspase-3 in all 3 groups are increased obviously. 3 days and 7 days after operation, the positive rates of caspase-3 in group C increased slowly. There is difference in the positive rates of caspase-3 between group A and other two groups(P<0.05).DiscussionSpinal cord injury may ultimately result in an irreversible and profound motor paresis due to dysfunction and loss of motor neurons at and around the level of spinal cord. Previous studies showed that deficiency of endogenous neurotrophins is associated with poor neuronal survival and even cell death. Thus, it is conceivable that injury of the spinal cord induces both neuronal regeneration, mediated by neurotrophins, and cell death. Brain derived neurotroph factor(BDNF) is an important member in the family of neurotrophic factors(NTFs). Its distribution in normal tissue of spinal cord demonstrates that it play an important role in development of spinal cord. After spinal cord injury, the level of BDNF and its expression of mRNA are increased, it can promote the survival,differentiation and outgrowth of motor neurons. Therefore, recent work is focusing attention on BDNF.Recent research showed the therapeutic effect of BDNF gene modified cell transplantation can become a new way in spinal cord injury. Bone marrow stromal cells(BMSCs) have become the ideal gene target cells for their advantages of multiple differentiations, amplification abilities and feasibility of transfection.In our study, The BDNF gene was introduced into BMSCs. The results suggest that the exogenous BDNF had been successfully transfected into rat BMSCs with liposomes-mediated transfection method. The stable expression of BDNF, which were validated at mRNA and protein levels, provide credible basis for further study.The therapeutic effect of BDNF-BMSCs transplantation in the spinal cord injury model was observed in this research. There were numerous cells in the local of injuried spinal cord which expressed the caspase-3. After spinal cord injury, the caspase-3 positive cells in group A and group B were decreased(P<0.05), it proved the BMSCs can prevent the cell necrosis, then protect the spinal cord cells. The number of caspase-3 positive cells in group C increased slightly, even 21 days after transplantation. Thus, cellular delivery of BDNF by BMSCs or hypersecreting BDNF-BMSCs may have a therapeutic effect following spinal cord injury through inhibiting the activation of caspase-3 and neuroprotective.BMSCs have been used in numerous preclinical models to regenerate a variety of injured tissues. Many researches have pay attention on the gene modify cell transplantation of spinal cord injury more and more. In this study, we explore the mechanism for BDNF-BMSCs to protect and repair the tissue in spinal cord injury. We also afford a new direct to therapy the spinal cord injury by gene modified cells transplantation safely and effectively.Conclusion1,BMSCs are capable of differentiating into neuronal cells by the induction of BDNF and extract of spinal cord. 2,The exogenous BDNF had been successfully transfected into rat BMSCs with liposomes-mediated transfection method.3,Transplantation of BDNF gene modified BMSCs can promote the survival and regeneration in spinal cord injury, it is more effective in repair of spinal cord injury than use BMSCs only.