| Objective To study the effect of 7,8-dihydroxyflavone（7,8-DHF）and Schwann cells（SCs）transplantation on axon regeneration and function recovery after acellular nerve allograft（ANA）repair of the sciatic nerve gap in mice.The effects of combined therapy on brain-derived neurotrophic factor（BDNF）,tyrosine kinase B（Trk b）and survival of transplanted SCS were examined,To clarify the effect of combined treatment on myelination,axon regeneration and functional reconstruction after sciatic nerve injury,and to clarify the mechanism of combined treatment to repair sciatic nerve injury,so as to provide a new combined treatment strategy for peripheral nerve injury.Methods The sciatic nerve cells were isolated and primary cultured,identified by S-100 immunofluorescence,and labeled by PKH26.Rat ana was prepared by chemical extraction.A total of 45 healthy SD rat were randomly divided into control group,Injection of 50μl PBS into ANA;SCs group:SCS（2×10~6/50μL）were labeled with PKH26 in ANA;and 7,8-DHF+SCs group:PKH26 labeled SCS（2×10~6/50μL）combined with 7,8-DHF（500 n Mol/place,2 places）was injected into ANA.ANA was used to bridge the defect between the two ends of 10 mm sciatic nerve,SFI and electrophysiology were used to evaluate functional recovery.Anesthesia was performed 28days after operation.BDNF and Trk B protein were evaluated by western bolt,observe the fluorescence signal of SCs labeled with PKH-26 by using immunofluorescence,observe the axonal regeneration and myelination by using NF,S100 immunofluorescence.Results1.After 8 and 12 days of primary culture,SCS showed fusiform or triangular cell bodies with good adherence to the wall.2.By double labeling of S-100 and DAPI,SCs positive cytoplasm was cultured,and the positive rate was 97.5%.3.The results of immunofluorescence staining showed that PKH26 labeled transplanted SCS could be seen in the middle of ANA in 7,8-dhf+SCS and SCS groups,No PKH26 labeled SCS was found in ANA group,The positive expression of PKH26 in7,8-dhf+SCS group was significantly higher than that in SCS group（P<0.001）.The myelination of ANA was labeled by S100 immunofluorescence,The results showed that S100 protein expression in middle segment of ANA in 7,8-DHF+SCS group and SCS group was significantly higher than that in ANA group,Moreover,S100 protein expression in 7,8-dhf+SCS group was higher than that in SCS group（P<0.001）.At the same time,the expression of NF protein in middle segment of ANA in 7,8-dhf+SCS group and SCS group was significantly higher than that in ANA group,The expression of NF protein in 7,8-dhf+SCS group was higher than that in SCS group（P<0.001）.4.The results of Western blot showed that the expression of BDNF and Trk B in SCS group and 7,8-dhf+SCS group was significantly higher than that in ANA group（P<0.001）.5.SFI test results:compared with Ana group,the SFI index of 7,8-dhf+SCS group and SCS group was significantly higher,and the SFI index of 7,8-dhf+SCS group was higher than that of SCS group（P<0.001）.6.Electrophysiological test results:The nerve conduction velocity and amplitude of SCS group and 7,8-dhf+SCS group were significantly higher than that of ANA group（P<0.05）,but the delay period was shorter than that of ANA group（P<0.05）,and the improvement of electrophysiological parameters of 7,8-dhf+SCS group was better than that of SCS group.ConclusionThe combined 7,8-DHF and SCs transplantation treatment promoted axonal regeneration,myelination and function recovery significantly more than SCs treatments,which may be related to 7,8-DHF upregulating BDNF and Trk B expression in the ANA,and promoting SCs survival... |
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