| Objective:Spinal cord injury(SCI)is a severe neuropathic disorder that causes sensory and motor dysfunctions.At present,cell transplantation for the treatment of SCI is a promising and popular research area.However,there are still some problems to overcome for cell transplantation,such as the low survival rate of cultured cells in vitro,the difficulty for cells to survive and transform in the in vivo inhibitory microenvironment,immunological rejection and ethical issues.In this study,we investigated whether different intensities of pulsed electromagnetic fields(PEMF)can optimize the biological functions of bone marrow mesenchymal stem cells(BMSCs)in vitro,and the effect of optimized BMSCs on the treatment of SCI was observed in vivo.Further,some potential biological mechanisms are discussed.Here,we explore a potential treatment option.Methods:1.In vitro experiments: BMSCs were extracted,isolated,purified and cultured from Wistar rats,and identified by flow cytometry.The activity and proliferation of BMSCs stimulated by PEMF with different intensities were observed.Then the optimal stimulation intensity was selected of 25,50,75 and 100 Hz PEMF(once a day,1 hour each time,1-9 days of continuous intervention).The activity and proliferative ability of BMSCs stimulated by PEMF of different intensity and duration were observed,and selecting the best stimulating intensity.Western-blot technique was used to observe the level of neurotrophic factors of BMSCs stimulated by PEMF of different intensity and duration.2.In vivo experiment: Spinal cord injury model of Wistar rats was established with Impactor model-? hitter.The rats were randomly divided into four groups: group A:experimental control group(treated with laminectomy alone);group B: injury alone group(treated with spinal cord injury modeling);group C: BMSCs transplantation group(treated with BMSCs without PEMF stimulation);group D: OBMSCs transplantation group(treated with high-activity BMSCs transplantation).BMSCs with high activity were transplanted into rats with spinal cord injury,and the fillingstatus,activity and proliferation ability of transplanted cells were observed by immunohistochemical method,and the effect of transplanted cells on local histological repair of spinal cord injury was observed.We also explored the correlation between PEMF and p75NTR-Trk signaling pathway after transplation of BMSCs,and evaluated the recovery of motor function in rats by BBB score.Statistical software SPSS 18.0 was used to analyze the data.The data were expressed as mean(MeanąSD).The significant difference was calculated by ANOVA testing,and P<0.05 was considered to have statistical significance.Results:1.This experiment completed the extraction,isolation,purification,culture and identification of BMSCs.The immunophenotypes of BMSCs were CD29,CD90 positive,CD34 and CD45 negative,which accorded with the phenotypic characteristics of BMSCs,and there was no cell contamination of hematopoietic system.2.The activity of BMSCs was significantly higher than that of 20 Hz,75Hz and100 Hz groups when PEMF was stimulated for 1 hour per day for 9 days.In this study,50 Hz,1h/d and 9d were used as the optimum stimulating intensity to prepare viable seed cells.WB test in vitro showed that the expression levels of BDNF and NGF in BMSCs stimulated by PEMF were significantly higher than those in the control groups(p<0.05),and NT-3 expression levels has an upward trend,and there was no significant difference between the two groups(p>0.05).3.In vivo experiments showed that the area of local syringomyelia after SCI injury in OBMSCs group was significantly smaller than control group(p<0.05),and the positive area of NF200 was significantly higher than control group(p<0.05),and the positive area of GFAP was significantly lower than control group(p<0.05).WB test results showed that the expression level of BDNF in OBMSCs group was significantly higher than control group(p<0.05).The expression level of NGF in OBMSCs group showed an upward trend,and there was no significant difference compared with the control group(p>0.05).The BBB function score in OBMSCs group was significantly higher than that in control group after 3 weeks of cell transplantation(p<0.05).WB test results showed that the expression levels ofp75 NTR,TrkA,TrkB and TrkC in OBMSCs group were significantly higher than those in control group(p<0.05).Conclusion:In this study,BMSCs were successfully isolated and cultured.The BMSCs expressed the highest biological activity and high levels of BDNF and NGF under stimulation intensity of 50 Hz,1 h/d and 9d of PEMF.In OBMSCs group,the formation of syringomyelia in the injured area of rats was significantly reduced,the survival of neurons and the expression of neurotrophic factors were promoted,the formation of glial scar was reduced,and the motor function of rats was significantly improved.In addition,the expression of p75 NTR,TrkA,TrkB and TrkC increased significantly in OBMSCs group,which may indicate the potential signaling pathway and regulatory mechanism of OBMSCs transplantation for SCI repair. |
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