| 【Objective】The recovery of neurological function after spinal cord injury(SCI)has always been a difficult problem for the medical community,which the axon regeneration and myelination is important way to break through this problem.Lentivirus-mediated Sonic Hedgehog(Shh)gene was used to transfect rat Schwann cells(SCs),continuous and stable secretion of Shh factor,and enhance the proliferation of SCs and promote the function of expressing neurotrophic factors.Transplanting genetically modified SCs to the site of SCI,and investigate the role of Shh combined with SCs in repairing SCI and its related mechanisms.The aim of this study is to provide new strategies for genetically modified cells to promote SCI repair.【Methods】1.Isolation and identification of rat Schwann cells: In this study,Sprague Dawley rats were selected from 3-5 days after birth,SCs were isolated from sciatic nerves.SCs were separated,cultured and purified by double enzyme digestion and differential attachment,and S-100 immunofluorescence was used to identify SCs.2.Lentivirus transfected Schwann cells and screening: Construction of p LVX-IRES-Puro-Shh Lentiviral Expression,passing the purified second-generation Schwann cells to plate and add virus working solution.After transfection,puromycin was added to get Schwann cells which stably expressing Shh factor.3.Detection of cell viability and expression factors: MTT assay for cell proliferation and viability,drawing the cell growth curve;multi-time point ELISA to detect changes in expression levels of Shh factor and BDNF,NGF.4.Establishment of spinal cord injury model and behavioral evaluation after transplantation: Female SD rat,8 weeks old,using Impactor Model II SCI System.Behavioral evaluations by BBB scores for each group of rats on the day after SCI and every 2 weeks within 12 weeks after SCI to assess the recovery of hindlimb function.5.The changes of nerve cells at the site of injury after transplantation: In the 12 th week after SCI,axons were marked with NF200,and the injury center was transected and stained to observe the changes of local axons in spinal cord injury;at the 2nd and 12 th week after SCI,Nestin-labeled neural stem cells,Olig2-labeled oligodendrocyte precursor cells,and GFAP-labeled astrocytes were transected and stained at the injury center to observe the proliferation and differentiation of neural cells.6.The changes of white matter and myelin sheath in the injured area after transplantation: At the 2nd and 12 th week after SCI,the rats in each group were transected with the injury site as the center,and stained with toluidine blue.The white matter and myelin sheath of the lesion were observed under high magnification.【Results】1.Observation of cell morphology and proliferation: The screened transfected Shh factor Schwann cells conformed to the typical characteristics of Schwann cells.S-100 staining observed that the transfected Shh factor Schwann cells were in the same morphology as normal cells,indicating that there was no burden on the cells after lentivirus transfection.After purification and culture,the growth of Schwann cells in each group was observed under light microscope.The cell density of the S-SCs group was higher than that of the SCs and V-SCs groups.2.Cell proliferation activity and expression factor detection: The cell proliferation curve was found to be exponential by MTT.Cell proliferation activity of the S-SCs group was higher than that of the V-SCs group and the SCs group(P <0.05);the contents of Shh,BDNF and NGF were detected by ELISA at different time points.The S-SCs group was higher than the SCs and V-SCs groups,and the difference was statistically significant.(P <0.01).3.Motor function evaluation: Animals were evaluated for BBB within 12 weeks after spinal cord injury,and hind limb function of each group of rats recovered.At 12 weeks after injury,compared with DMEM group and SCs group,S-SCs group BBB score was the highest(P <0.01).The S-SCs group better promoted the recovery of the motor function of the hind limbs of rats.4.Changes of nerve cells at the site of injury: The results showed that compared with the SCs group and the DMEM group,the number of NF200 positive cells in injury center of the S-SCs group was the largest(P <0.01);in the 2 and 12 week after SCI,the results showed that the number of Nestin-positive cells in injury center,Olig2-positive cells in injury center and surrounding white matter was the most in the S-SCs group;in the second week after injury,the number of GFAP-positive cells in injured area was more in DMEM group than SCs group and S-SCs group(P <0.05).The number of GFAP-positive cells in the SCs group was the least(P <0.01).5.The changes of white matter and myelin sheath in the injured area after transplantation: At the 2nd week of the SCI,the white matter area of the SCI center in each group was damaged;at the 12 th week,the nerve fiber structure of the white matter area in DMEM group was unclear and disorderly arranged;while in S-SCs group,the nerve fiber structure of the white matter area was clear and the cystic structure was visible The presence of myelin sheath can improve the early injury area of SCI and reduce the formation of cavities in the chronic phase compared with the DMEM group.【Conclusions】1.Lentivirus-mediated Shh factor-transfected Schwann cells can stably and continuously express Shh factor,can promote the proliferation of Schwann cells and the expression of neurotrophic factors BDNF and NGF,and its effect has long-term stable characteristics.2.Local transplantation of spinal cord injury sustained and stable expression of Shh factor cells,compared with SCs or DMEM alone,significantly reduce the formation of glial scars in injured area,promote axon regeneration and myelinization in the injured area,and promote the recovery of hind limb function in rats.3.Shh factor can enhance the self-activity of SCs,continuously and stably express Shh.The two are combined through genetic modification to produce a synergistic effect,activate endogenous neural cells,and promote the direction of neural stem cells to oligodendrocytes.Differentiation,protection of axons and promotion of remyelination of axons provide a new strategy for genetic modification combined with cell transplantation to repair spinal cord injury. |
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