A Study On Spinal Cord Injury Repair By Transplantation Of Modified Activated Schwann Cells With SHH Gene

【Objective】To obtain activated schwann cells(ASCs)enhanced by expressed sonic hedgehog(SHH)and to preliminarily explore the synergistic effect between ASCs and sonic hedgehog to repair spinal cord injury【Methods】Cell experiment in vitroSciatic nerve from 4-to 6-day old SD rats was pre-ligated to get active SCs.pEGFP-N1-SHH was transfected into ASCs by liposome transfection,and GFP positive cells were sorted by flow cytometry.Cell proliferation was observed by EdU staining and expressions of SHH,BDNF and NGF were determined by ELISA.Group I,the non-activated SCs group;Group II,the un-transfected ASCs group;Group III,the blank plasmid-transfected ASCs group;Group IV,the SHH-expressing plasmid transfected ASCs group.Transplantation treatment in vivoFemale SD rats aged 8-10 weeks were selected and T10 spinal cord injury model was established using the IMPACTOR MODEL-II SCI device.Group A,the DMEM transplant group,Group B,the ASCs transplant group,and Group C,the S-ASCs transplant group;The prepared cells were transplanted into the lesions 1 week after SCI and the functional recovery was evaluated using BBB score system;With the lesion as the center,spinal cord segments were removed from animals and expression of SHH was determined by ELISA;NF200 immunofluorescence staining was conducted to evaluate local axonal change,and Nestin,Olig2 and GFAP immunofluorescence staining were conducted to evaluate the proliferation and differentiation of endogenous neural cells;Luxol fast blue staining was carried out in SCI center,and the area ratio of white matter and myelin sheath were calculated.【Results】Cell experiment in vitroEdU staining showed the highest positive rate in Group IV(26.17±4.03%),followed by Group II(20.41±3.16%)and Group III(19.34±2.82%),and the lowest in Group I(11.86±2.98%)(P<0.05);The expression levels of SHH,BDNF and NGF in Group II,Group III,and Group IV at different time points increased compared with that of Group I,among which the highest levels were observed in Group IV(P<0.05).Transplantation treatment in vivoThe function of hind limbs of rats in all groups recovered from 2 to 12 weeks after SCI.BBB scores of Group C,Group B and Group A at 12 weeks after SCI decreased successively(P<0.01);Local expression of SHH in Group C was the highest among 3 groups at 2 and 4 weeks after SCI(P<0.01);At 12 weeks after SCI,NF200 positive expression in SCI center was counted,revealing the most NF200 in the group C(55.13±12.16 points/field),followed by the group B(23.63±8.75points/field)and then the group A(10.50±5.58 points/field)(P<0.01);At 2 and 12weeks after SCI,Nestin~+cells and Olig2~+cells in SCI center reduced in the group C,group B and group A successively(P<0.05),but showed no obvious changes with time(P?0.05);At 2 weeks after SCI,GFAP~+cells in SCI center of the group B and the group C were more than those of the group A(P<0.01),and at 12 weeks after SCI,the group C presented the least GFAP~+cells(P<0.01);At 4 and 12 weeks after SCI,LFB staining demonstrated a stable ratio of white matter(P?0.05)but an increased area of myelin sheath with time(P?0.05).At each time points,the ratio of white matter was the highest and the area of myelin sheath was the largest in group C,followed by group B and then group A(P<0.05).【Conclusions】1.Liposome mediated SHH-expressing plasmid transfection enhances the activation of ASCs and promotes the expression of SHH,BDNF and NGF.The enhancement of ASCs activation state can not maintain stably.2.Compared with intensively studied ASCs transplantation,local transplantation of ASCs enhanced by the transfection of the Shh gene in the subacute stage can contribute more to the recovery of hind limb function.The present animal experiment has preliminarily confirmed the effectiveness of SHH combined with Schwann cell transplantation for SCI repair.3.After SCI,ASCs act as a carrier for the local short-term expression of SHH and SHH can enhance ASCs in turn.The combination of ASCs and SHH can provide exogenous transplanted cells,activate endogenous transplanted cells,increase neural stem cells and oligodendrocytes precursor cells.The timing sequence cooperation between ASCs and SHH can promote axonal myelination,protect axones and further support the relevance between myelination and functional recovery.