| BackgroundSpinal cord injury (SCI) is one of the common neurosurgical emergency crisis, the international medical problems in recent years with the development of transportation and construction industry, the incidence of spinal cord injury was also found increased year by year. Spinal cord injury with high morbidity and low cure rate characteristics, often left paraplegic after injury, incontinence, difficulty walking and other neurological disorders, and therefore also lost the ability to work, and even self-care ability, not only to the patients themselves huge pain, but also a huge burden to the family social state. Nerve regeneration is also one of the problems plagued the medical profession. Previous studies showed that the damaged area is extremely detrimental to the local microenvironment and glial scars are nerve regeneration obstacles. Neurotrophic factors in nerve regeneration and repair process has been playing a pivotal role, neurotrophic factor -3 (NT-3) is one of the important neurotrophic factor family member, lack of damage to the region’s own secretion of trophic factors, and also the presence of neurotrophic factor inhibiting factors play a role, the researchers showed that cytokine inhibitory protein 3 (SOCS3) is a key molecule inhibition of endogenous neurotrophic factors, leading to axonal regeneration difficult. Single treatment approach limited role to promote recovery of neurological function, combination therapy is likely to break the nerve regeneration strategies using cell transplantation constant supply of exogenous neurotrophic factor to improve the local damage unfavorable microenvironment, while application of gene silencing suppression hinder neurotrophic factors play a role in the factors that combined endogenous and exogenous neurotrophic factor treatment of spinal cord injury, spinal cord injury is expected to bring a breakthrough.ObjectiveThe United siRNA technology neurotrophic factor inhibiting factors that hinder the role and use of gene transfer technology to improve the microenvironment of the damaged area, in order to achieve the purpose of promoting nerve regeneration.Methods① Adherence method in vitro human umbilical cord-derived mesenchymal cells (HUMSC), while the separation, purification and identification.② Construction of NT-3 gene eukaryotic expression vector, using gene transfection technology into its HUMSC, constructed NT-3-HUMSC cell survival in vitro assay conditions and NT-3 expression.③ The role of literature in SOCS3 screening of specific targets, for sequence homology analysis, the establishment of a negative control, the design and synthesis of siRNA, while in vitro detection.④ SD rats with spinal cord injury model established intol. sham group 10; 2.T12 whole spinal cord injury model 40, were randomLy divided into four groups, respectively; saline treatment group 10; siRNA+ NT-3-HUMSCs treatment group 10; NT-3-HUMSCs treatment group 10; siRNA treated group 10. For the sham group, open only lamina, exposing the spinal cord, but do not damage the spinal cord. Other experimental group after 6 weeks transection local injection of siRNA,7 weeks after retransplantation NT-3-HUMSCs, HUMSCs or NT-3. After each group above modeling success, respectively in January and February and March survival motor function evaluation and neurophysiological monitoring.⑤ Pairs of SD rats were perfusion fixed and drawn, observe local glial scar degradation and axonal regeneration, while the use of RT-PCR and Western blot NT-3 protein expression.The results:1) In vitro constructed by NT-3-HUMSCs neurotrophin 3 expression was significantly increased compared with HUMSCs and NT-3 promotes the induction of human umbilical cord matrix cells towards neuronal differentiation.2) SOCS3-siRNA+ NT-3-HUMSCs SD rats with spinal cord injury combined treatment group was significantly higher than BBB scores NT-3-HUMSCs and SOCS3-siRNA and sham groups. The difference was statistically significant (p<0.05)3) SOCS3-siRNA + NT-3-HUMSCs combined treatment of spinal cord injury group SD rats crawl grid experiments showed neurological recovery was significantly higher than that of NT-3-HUMSCs and SOCS3-siRNA and sham groups. The difference was statistically significant (p<0.05).4) siRNA+ NT-3-HUMSCs treatment group compared with saline treated group was significantly reduced transection syringomyelia (p<0.05), a significant difference5) Tracking results show iRNA+NT-3-HUMSCs treatment group compared with saline treated axons grow significantly.6) Electrophysiological testing nerve damage after 12 weeks of treatment group compared with shorter incubation period P40 sham group, siRNA+ NT-3-HUMSCs treatment group compared with saline treatment group was significantly shorter incubation period, volatility increased significantly, the difference was statistically significant (p<0.05).ConclusionNT-3-HUMSCs joint SOCS3 gene silencing can improve exogenous NT-3 expression and increased inhibition of endogenous SOCS3 NT-3 utilization, improve the local micro-environment, so as to promote nerve repair damage. |
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