Study On Influence Of Vitrified Cryopreservation Contain Ligustrazine On Treating Defect Of Sciatic Nerve In Rat With Tissue-engineered Nerve

Objective: To study the function and mechanism of transplanting with allograftnerve cryo-preserved by vitrified cryopreservation of ligustrazine fluidcombined with NSCs treating long defect of sciatic nerve inrat.Methods:(1)Neural stem cells(NSCs) were isolated, cultured,purified fromnewborn rats cerebral cortex and NSCs were transfected by AdenovirusContaining Green Fluorescent Protein(AD-GFP).（2）48sciatic nerves withlength of1.5cm,obtained from24adult Wistar rats are divided into tow groupsrandomly.One group is cryo-preserved at-196℃for3weeks with vitrifiedcryopreservation containing ligustrazine fluid(provided for group C)．Onegroup is cryo-preserved at-196℃for3weeks with vitrified cryopreservationwithout ligustrazine fluid(provided for group B).(3)72adult SD rats(male andfemale are unlimited) were established to be the model of right side1.5cmsciatic nerve defect and then divided into A,B,C group randomly,24rats pergroup.C group (experimental group): cryopreserved rat sciatic nerve wereseamed end to end in the rat sciatic nerve defect model,and inject the genemodified NSCs under the epineurium. Group A (control group): we seam thereversal autologous nerve end to end with the model of1.5cm length sciaticnerve defect and inject the same volume of normal saline.Group B (control group):we seam the cryo-preserved nerve end to end with the model of1.5cmlength sciatic nerve defect and inject the same volume of neural stem cells’liquid growth medium.(4)sciatic nerve function index (SFI),Sciatic nerveconduction velocity（NCV），gastrocnemius muscle wet weight ratio (woundedside/health side),Area proportion of nerve myelin sheath (sem) were valuated at2,4,8,12weeks after operation.(5)The data were analyzed by SPSS19.0software and IPP software. One way ANOVA and LSD were used to analyzethe difference between groups. The result was described as, x±s. P<0.05indicated the difference was statistically significant.Results: Observation at12weeks after operation indicated: SFI group A-64.89±4.76,group B-73.22±3.21,group C-69.31±2.13,F=9.36,p=0.002,group A>group C> group B, andthe difference is with statistically significant (P <0.05). NCV: group A19.72±1.61,group B16.91±0.48， group C18.27±0.78,F=10.36， p=0.001.groupA>group C> group B,and the difference is statistically significant (P <0.05).gastrocnemius muscle wet weight ratio (wounded side/health side): groupA0.68±0.08,group B0.57±0.05,group C0.65±0.05,group A,group C> groupB, and the difference is statistically significant (P <0.05)，there is no significantdifference between group A and C. Myelin sheath thickness under electronmicroscopy:group A45.66±1.28， group B36.47±1.33， group C41.85±1.39,F=71.58，p=0.00.group A>group C>group B, and the difference wasstatistically significant (P <0.05). Area proportion of nerve myelin sheath(sem): group A45.66±1.28， group B36.47±1.33， group C41.85± 1.39,F=71.58，p=0.00.group A>group C>group B,and the difference wasstatistically significant (P <0.05). From the appearance of transplanted nerveperiod:group A and group C transplanted nerve is closer to the originaldiameter of sciatic nerve than group B, and blood vessels are observed acrossthe nerve bridge.there are no significant difference between the groups of2,4,8weeks after operation. Conclusion:1,A large number of NSCs can becollected,cultivated and go down to the future generation from the new bone ratcerebral cortex.NSCs can differentiate into a variety of function cells in the caseof bFGF,EGF,B27were consumed slowly;2, NSCs can coexist with allograftsciatic nerve cryo-preserved by vitrified cryopreservation of ligustrazine fluidinside the receptor rats. NSCs can differentiate into nerve cells and glial cellsand restrain muscle atrophy;3, It is a effective way that allograft nerve in ratscryo-preserved by vitrified cryopreservation of ligustrazine fluid combined withNSCs transplant to treat long defect of sciatic nerve in rat.