| Objective:Peripheral nerve injury is a common and frequently-occurring disease in orthopedic clinical work.The most common is the nerve damage directly caused by trauma.The peripheral nervous system has endogenous repair and regeneration potential.Severe peripheral nerve rupture leads to axonal degeneration and central neuronal death.It is still challenging to achieve local nerve tissue regeneration and functional reconstruction.After nerve injury,directly microsurgical suture therapy is a commonly used treatment;when the nerve defect segment is too long,autologous nerve graft or artificial nerve conduit bridging repair is needed.Peripheral nerve repair involves a complex series of cellular and molecular regulation processes,and simple surgical repair does not solve all problems.Although the peripheral nerves of the injury have achieved local reconnection,residual nerve pain and weak recovery of distal nerve function have profound effects on the individual,which brings great difficulties to normal work and life.Current stem cell therapy is widely used in the field of regenerative medicine,and the application of stem cells in peripheral nerve injury also shows great potential.In recent years,many studies have shown that brain dopaminergic neurotrophic factor(CDNF)has neuroprotective effects in the midbrain;in the rat sciatic nerve injury model,direct expression of CDNF by a lentiviral vector in nerve catheter bridging site can improve the function of lower limbs in rats.The single application of any current method cannot meet the requirements of damaged nerve function reconstruction,and combination therapy provides a new direction for nerve regeneration.Research purposes:In this study,rat bone marrow mesenchymal stem cells(MSCs)were first extracted and identified,and transfected with a lentiviral vector to establish a cell vector capable of continuously expressing CDNF.Local application of stem cell therapy vector in rat sciatic nerve bridging model,we explored the therapeutic effect of rat MSCs transfected with CDNF in combination with artificial collagen catheter on local nerve regeneration,survival of central neurons and distal target organs to evaluate the impact of the combination therapy on peripheral nerve regeneration.Methods:In vitro experiments(1)Identification of MSCs The bilateral femurs of adult rats were taken and the MSCs of the rats were extracted with the marrow cavity.The cultured third generation cells were used to identify the expression of CD29 and CD90 by flow cytometry.(2)Transfection and detection of lentiviral vectors The MSCs in well-grown 3rd generation were transfected with a lentiviral vector containing CDNF.The state of the transfected cells was observed and cultured for 2 weeks.The protein expression of CDNF in MSCs was detected by Western blot.In vivo experiments(1)Rat neural sciatic nerve defect model was made Rat MSCs transfected with CDNF lentiviral vector were used in nerve injury models to observe the effect of collagen catheter combined with CDNF on nerve regeneration and repair.(2)Detecting the expression of CDNF in sciatic nerve of rats After 4 weeks,the sciatic nerve 1cm distal to the bridging area was taken and stored in liquid nitrogen.The expression of CDNF in each group was detected by Western blot.(3)Retrograde tracing sciatic nerveAfter 12 weeks,4 rats in each group were randomly selected and 3 normal rats were added as a control.Horseradish peroxidase(HRP)was injected distal to the nerve bridging area for retrograde tracing.Regeneration effect evaluation(1)Histological analysis of newborn nerveImmunohistochemical(1HC)technique was used to stain S100 and NF200 in the regenerative segment of the nerve to show the regeneration of myelin and axon in the bridging region.(2)Analysis of the local structure of the catheter and regenerative nerve by Electron microscopyAt 8,12 weeks after surgery,scanning electron microscopy was used to observe the artificial collagen catheters and monitor the catheter degradation in vivo;transmission electron microscopy was used to observe the regeneration segment of the sciatic nerve and quantitative analysis the number of axon regeneration and thickness of medullary sheath.(3)Counting of central surviving neuronsStatistical analysis was performed on the horseradish peroxidase-positive neurons after staining with L4-5 in rat spinal cord.(4)Sciatic nerve index testAt 2,4,6,8,10 and 12 weeks after the operation,rats were recorded in gait and the rat footprints were analyzed to calculate the sciatic nerve index(SFI).(5)Recovery of gastrocnemius musclesAt 4,8 weeks after surgery,gastrocnemius muscle on the operation side and the normal side were taken to weigh to calculate the muscle ratio.Recovery of gastrocnemius muscle was detected by Masson staining.Results:(1)Co-expression of CD29 and CD90 in MSCsCDNF protein expression was increased in MSCs infected with recombinant CDNF lentiviral vector after 2 weeks culture,while no CDNF protein is detected in the control group.(2)At 4 weeks after the operation,the expression of CDNF protein was increased in CDNF-MSCs application group compared with that of the LV-MSCs,MSCs and DMEM groups.(3)At 4,8 and 12 weeks,scanning electron microscopy showed that collagen nerve catheter gradually degenerated in rats and the thickness of the tube wall became thinner with time.(4)At 4,8 and 12 weeks after surgery,better nerve regeneration was observed morphologically in the CDNF-MSCs group than that in the LV-MSCs,MSCs and naive groups tested by S100 and NF200IHC.(5)At 8 and 12 weeks after surgery,axillary diameter and myelin sheath sciatic nerve index of CDNF-MSCs group showed good myelin maturation under transmission electron microscopy.(6)Sciatic nerve index analysis showed that the neurological function of the hindlimbs of rats recovered obviously in the application group of CDNF-MSCs.(7)The results of retrograde tracing of nerve by horseradish peroxidase showed that the application of CDNF-MSCs promoted the survival of central neurons after injury.(8)At 8 weeks and 12 weeks after the operation,the wet weight of muscle in the CDNF-MSCs group was increased obviously compared with that in the no repair group,LV-MSCs group,MSCs group and DMEM group.The muscle wet weight ratio of CDNF-MSCs group was significantly higher than that of No repair group,LV-MSCs group,MSCs group and DMEM group.Conclusion:Our study demonstrates that intracavitary use of MSCs that bind to recombinant CDNF lentiviral vectors has multiple therapeutic effects:(1)localized lesions of axons and myelin can be promoted locally,and myelin maturity is improved.(2)protecting the survival of central motor neurons in the spinal cord;(3)significantly reducing the atrophy of the gastrocnemius in the distal target of the injured nerve;(4)significantly improving the recovery of the lower limb motor in rats.The combination of nerve conduit with CDNF gene therapy and cell therapy provides a new therapeutic strategy for the treatment of peripheral nerve injury. |
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