The Study On Spinal Cord Injury Repaire By Schwann Cells Stimulated With Low-intensity Pulsed Ultrasound

[Objective]Spinal cord injury(SCI)is a devastating neurotraumatic disease.Improving the local microenvironment after spinal cord injury to promote axonal regeneration and and neural function recovery has become an important direction of current research.Neurotrophic factors(NTFs)are important protein molecule that regulates many pathophysiological processes in unbalanced microenvironment after spinal cord injury.Neurotrophic factors play an irreplaceable role in protecting cell survival,extenuating inflammatory responses,promoting axon regeneration and remyelination.Studies have shown that Schwann cells(SCs)promote the recovery of neurological function by secreting many kinds of neurotrophic factors,such as BDNF,NT-3,NGF,FGF,GDNF and so on.Previous studies have shown that low-intensity pulsed ultrasound(LIPUS)could promote the secretion of neurotrophic factors in bone marrow mesenchymal stem cells,neural stem cells and other cells.In this study,LIPUS was used to stimulate Schwann cells to promote the secretion of neurotrophic factors in vitro.The transplantation of Schwann cells stimulated with LIPUS plays a neuroprotective and inflammatory regulatory role,and promotes axonal regeneration and motor function recovery in rats with spinal cord injury,which provides a new method for the combined therapy of cell transplantation in spinal cord injury.[Methods]This experiment consists of two parts:1.Isolation and culture of Schwann cells and activated Schwann cells(pre-degenerated for 7 days);To observe the changes of biological characteristics of LIPUS-stimulated Schwann cells(LIPUS-SCs)in neurotrophin secretion,cell proliferation,apoptosis and migration,and compare with activated Schwann cells.2.To observe the improvement of microenvironment,glial scar formation,axon growth and remyelination after LIPUS-SCs transplantation,and to explore the mechanism of LIPUS-SCs transplantation in promoting axonal regeneration and repairing spinal cord injury through NRG1/ErbB signal pathway.In vitro:1.Isolation,purification and identification of Schwann cells and activated Schwann cells:Schwann cells and activated Schwann cells were extracted from the sciatic nerve of 4-week-old female Wistar rats(unilateral pre-degenerated for 7 days).The cells were cultured and purified by double enzyme digestion and differential adhesion method.The purity of the cells was identified by S-100 staining.2.Establishment of LIPUS stimulation system and experimental grouping:the Schwann cells purified to the third generation were stimulated with an intensity of50m W/cm~2,5 minutes for 3 days.This experiment was divided into three groups:SC group;LIPUS-SCs group;and ASCs group.3.Related detection of biological characteristics of Schwann cells regulated by LIPUS:the expression levels of BDNF,NT-3 and NGF in the supernatant were detected by ELISA method;CCK-8 method was used to detect Schwann cell proliferation activity;Annexin V-FITC/PI staining was used to detect Schwann cell apoptosis;Transwell migration test was used to evaluate Schwann cell migration in vitro.The regulatory effect of LIPUS on Schwann cells was comprehensively evaluated and compared with ASCs group.In vivo:1.Establishment of an animal model of SCI:adult female wistar rats(n=60)were divided into 4 groups:Sham group,Injury group;SCs transplantation group;and LIPUS-SCs transplantation group.Cell transplantation was performed on the 7th day after spinal cord injury.2.Behavioral evaluation of motor function recovery:the rats in each group were scored by BBB once a week until the end of the 8th week.3.Detection of changes of neurotrophic factors and inflammatory factors:detection of BDNF,NT-3 and NGF in spinal cord tissue by ELISA method.The changes of IL-1βand IL-10 were detected by Western blot.4.Detection of tissue sparing after injury:the spinal cord tissue of rats was taken for HE staining to observe the tissue sparing in each groups.5.Detection of axon regeneration and astrocyte activation:neurofilament protein-200(NF200),glial fibrillary acidic protein(GFAP)and DAPI staining were performed on the spinal cord sections at 8 weeks after spinal cord injury.The axon growth,astrocyte activation and glial scar formation after cell transplantation were observed.6.Detection of the changes of NRG1/ErbB signal pathway:the expressions of NRG1,ErbB2 and MBP in spinal cord tissue of each group were detected by Western blot.[Results]1.Schwann cells and activated Schwann cells were successfully isolated and purified(pre-degenerated for 7 days).The purity of Schwann cells was more than 95%by S-100 fluorescence identification.2.LIPUS stimulation promoted the expression of BDNF,NT-3 and NGF in Schwann cells:the expression of BDNF,NT-3 and NGF in LIPUS-SCs group was significantly higher in SCs group(P<0.01),and similar to that in ASCs group(P＞0.05).3.LIPUS stimulation increased the proliferation activity of Schwann cells:compared with the SCs group,the cell proliferation activity of LIPUS-SCs group increased significantly(114.1%±1.754%,P<0.01).Compared with ASCs group(112.5%±2.032%),the cell activity of LIPUS-SCs group was higher than that of ASCs group(P<0.05).4.LIPUS stimulation is a safe physical intervention method:Annexin V-FITC/PI staining showed that the apoptosis rate of Schwann cells stimulated with LIPUS was lower than that of normal Schwann cells(2.003%±0.4888%vs.1.447%±0.1050%,P＞0.05),LIPUS stimulation could not induce apoptosis.5.LIPUS promotes Schwann cell migration:the number of Schwann cells stained by crystal violet in LIPUS-SCs group(53.5±4.203/visual field)was significantly higher in SCs group(31±4.761/visual field).More crystal violet staining cells were also observed in the ASCs group(57.50±6.557/visual field),which was not significantly different from that in the LIPUS-SCs group.6.LIPUS-SCs cell transplantation promoted the recovery of motor function:the hindlimb function of rats recovered at the 8th week after cell transplantation.At the end of the 8th week,the BBB score was the highest in the LIPUS-SCs transplantation group(15.667±0.578),the second in the SCs transplantation group(14±0)and the lowest in the injury group(6.333±0.578).7.LIPUS-SCs transplantation promoted the expression of local neurotrophic factors and improved inflammatory response:ELISA detected the secretion of neurotrophic factors in spinal cord injury 2 weeks after injury.The results showed that the expression of BDNF,NT-3 and NGF in spinal cord was the highest in LIPUS-SCs transplantation group,which was better than that in SCs transplantation group(P<0.01).LIPUS-SCs transplantation also has effect of inflammatory regulation,which can inhibit the overexpression of pro-inflammatory factor IL-1βand promote the expression of anti-inflammatory factor IL-10.8.LIPUS-SCs transplantation promotes the recovery of nerve function:HE staining shows that cell transplantation,especially LIPUS-SCs transplantation preserve tissue morphology after injury and reduce the formation of syringomyelia and infiltration of inflammatory cells.NF200/GFAP immunofluorescence staining showed that compared with Injury group,more NF200 labeled axons were observed in SCs and LIPUS-SCs cell transplantation groups,while GFAP immunofluorescence intensity was significantly lower than that in Injury group,and the number and average length of NF200 positive axons in LIPUS-SCs transplantation group were significantly higher than those in SCs transplantation group.9.LIPUS-SCs transplantation promotes axonal regeneration and repair of spinal cord injury by activating NRG1/ErbB signal pathway:the proteins expression of NRG1/ErbB signal pathway were detected by Western blot.The results showed that the protein expression levels of NRG1,ErbB2 and MBP in LIPUS-SCs group were significantly higher than those in SCs group.[Conclusions]1.Schwann cells stimulated with LIPUS(LIPUS-SCs)could significantly increase the expression of BDNF,NT-3 and NGF,promote cell migration and cell proliferation activity,and will not trigger cell apoptosis.LIPUS is a safe physical intervention.2.Compared with activated Schwann cells obtained by pre-degenerated,LIPUS-SCs has similar ability of neurotrophic factor secretion and cell migration,which provides a new method for efficient acquisition of Schwann cells.3.LIPUS-SCs cell transplantation can promote the secretion of local nutritional factors,regulate inflammatory response,inhibit the proliferation of reactive astrocytes,contribute to axon regeneration and axonal growth after spinal cord injury,and obtain better motor function recovery.4.LIPUs-SCs cell transplantation promotes axonal regeneration and repair of spinal cord injury by activating NRG1/ErbB signal pathway.