| 【Objective】The aim of this study was to realize the early intervention for muscle atrophy after spinal cord injury by using exsomes of Schwann cells and to explore the main mechanism,which provides a theoretical basis for the repair of muscle atrophy after spinal cord injury.1.To compare different types of muscle atrophy through proteomics analysis of atrophic muscle after spinal cord injury by 2D glue,and clarified the molecular basis of muscle atrophy after spinal cord injury;2.To elucidate the biological and molecular basis of the intervention of Schwann cells exsomes for muscle atrophy through construct the co-culture system of Schwann cell exosomes and L6 cells in vitro;3.To study the changes of important molecules,cells and tissues by local transplantation of Schwann cells exsomes,and to clarify the repair effect of exsomes of Schwann cells on muscle atrophy after spinal cord injury;4.To explore the potential mechanism of Schwann cells exsomes in repairing muscle atrophy after spinal cord injury through verifying the role of miR-206 and Akt / mTOR signaling in the exsomes treatment for atrophic muscle fibers after spinal cord injury.【Methods】1.The preparation of NYU spinal cord contusion model of rat,2D glue for protein separation,and protein mass spectrometry analysis,and bioinformatics analysis.2.Isolation,culture and identification of Schwann cell.Electro microscopic was used to identify the exsomes of Schwann cells,and Western Blot was used to identify the surface markers of Schwann cell.The culture and differentiation of L6 cell,and the dexamethasone induced atrophy model was established.Exsomes of Schwann cells was marked by PKH67,and the expriment of L6 cells to internalize exsomes,immunofluorescence staining and Gill’s H&E staining were also conducted.The expression of atrogin-1 and MuRF1 was detected by RT-PCR.3.Local injection of Schwann exsomes in lower limb gastrocnemius muscle.The size of muscle fibers was detected by H & E staining.The regeneration of muscle fiber was measured by Pax7 and MyoD1 immunofluorescence.Apoptosis was assed by TUNEL staining.Lower limb function was assessed by BBB score(Basso,Beattie & Bresnahan locomotor rating scale).The degree of injury was evaluated by the area of injured spinal cord.Fibrous scar area was measured through GFAP staining.Axonal regeneration was determined by BDA tracer.4.The activation of Akt/mTOR signaling pathway was detected by Western Blot after the intervention of Schwann cell exosomes.RT-PCR was used to monitor the effect of miR-206 on muscle atrophy after spinal cord injury and the change of miR-206 after Schwann cell exosomes applied.【Results】1.SCI reduced soleus muscle diameter.The average diameter of the soleus muscle of the control group was 94.4 ± 10.8 μm.In the SCI group,on day 7 and 14,the diameter of the soleus muscle fibers was 76.7 ± 10.0 μm and 66.2 ± 8.2 μm.The diameter of the soleus muscle fibers was significantly lower than that of the control froup(P <0.01)after spinal cord injury.The mean diameter of the soleus muscle and muscle fibers were gradually decreased at different time points.There were >50 proteins were changed in SCI rats compared with the control group.There were 23 protein spots showed significant changes of all changed proteins,and these proteins were sufficient to be identified by MALDI-TOF by the quantity.Use Mascot to analyze MS data search engines.Twenty different protein spots were identified.Confidence score of proteins > 95%.The identified proteins were identified into nine functional category databases by using PANTHER,including bioadhesive,cellular component or biogenesis,cell processes,developmental processes,immune system processes,localization,metabolic processes,multicellular biological processes,and response stimuli.2.The Schwann cell culture system was successfully constructed in vitro.The purity of Schwann cells was up to 98% by S100 and DAPI staining.The exsomes were extracted by cryogenic ultracentrifugation.Schwann cell markers: CD63,TSG101,Hsp70,Hsp90 and Flot-1 were positive,while the nuclear protein TFIIB was negative.The expression of CD63,TSG101,Hsp70,Hsp90 and Flot-1 were positive.Schwann exsomes labeled by PKH67 confirmed that can be L6 cells uptake;after the formation of L6 myotubes,the dexamethasone(Dex)was used to induced atrophy,and the diameter and area of myotubes showed significant improvement compared with the group used conditioned medium without exsomes and control group(P <0.05).The expressions of Atrogin-1 and MuRF were significantly down-regulated in the treatment group,and the expression of Atrogin-1 and MuRF were significantly lower than that of the control group(P<0.05),and in vitro experiments have shown that repair is effective.3.Schwann cell local injection in rat hind limb after spinal cord injury.Cross-sectional area of gastrocnemius was significantly improved(p<0.05)than the simple spinal cord injury group and the group used Schwann cell conditioned medium without exsomes(P <0.05),The tibialis anterior muscle and the proportion of soleus in different periods obtained different degree of recovery.The number of Pax7 + / MyoD1 + double positive cells in the muscle tissue of the exsomes group was significantly higher than that in the control group(P<0.05)after injury.In exsomes group,the expression of Foxo3,Atrogin-1,MuRF were reduced(P<0.05),the apoptotic rate of muscle fibers in the exoskeletal group was significantly lower assessed by TUNEL staining(P<0.05).The results showed that the extracorporeal mass of Schwann cells could promote the recovery of spinal cord injury after spinal cord injury,which suggested Schwann exsomes could promote the conversion restore between fast and slow muscle after spinal cord injury.BBB score showed that the hind limb function of the exsomes group was higher and the recovery rate was higher(P<0.05).There was no difference in spinal cord injury area,GFAP positive area and BDA tracer experiment among these groups,suggesting that functional recovery was related to muscular atrophy but spinal cord nerve recovery.4.RT-PCR was used to detect the miR-206 level inmyofiber of rats with spinal cord injury and sciatic nerve injury.It was found that miR-206 decreased significantly in the muscle fibers after spinal cord injury.The extent of Akt and mTOR phosphorylation in the gastrocnemius muscle was improved in exsomes group,suggesting that the anti-atrophy effect was related to the activation of Akt/mTOR signaling pathway,which suggested that exsomes of Schwann cells repair muscle atrophy after spinal cord injury was mediated by miR-206 and Akt/mTOR signaling pathway.【Conclusions】After the spinal cord injury,myofibrillar proteins were changed early after spinal cord injury,and muscle atrophy after spinal cord injury has its specificity compared with other muscle atrophy.Schwann exsomes could effectively inhibit L6 muscle atrophy and inhibit the expression of genes related to atrophy;Schwann exsomes could reduce the muscle atrophy after spinal cord injury and can promote the functional recovery of spinal cord injury;Schwann exsomes may suppress muscle atrophy after spinal cord injury through the up regulation of miR-206 and activation of Akt/mTOR signal pathway. |
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