Preclinical Study Of Standardized Activated Schwann Cells In Repairing Of Spinal Cord Injury

?Objective?The aim of this study was to obtain a model of efficient activation of Schwann Cells(SCs)and high expression of trophic factors in nonhuman primates,and to establish a model of standardized non-human primate Spinal Cord Injury(SCI),thus to repair primate SCI by the highly active activated Schwann cells transplantation.In order to standardize the evaluation standard of transplantation for repairing SCI,behavioral scoring,sensory evoked potential(SEP),motor evoked potential(MEP)detection and histology observation were carried out to determine the proper transplantation time and transplantation concentration of the activated Schwann cells to repair SCI.?Method?1.Double lower limb sural nerve of cynomolgus monkey were ligatured 7days before the culturing.The Schwann cells were purified by trypsin and collagenase and were identified by S100 immunofluorescence staining after 5 days in vitro.nucleus were marked by DAPI staining to detect the purity of SCs.2.The expression of myelin protein in SCs was detected by RT-qPCR and Western blotting after the addition of recombinant interleukin-11(IL-11).3.A model of SCI in nonhuman primates was made using SS-II large animal spinal cord machine(hit head diameter 5mm,striking impulse 50 g × 50mm).After the model was completed,the function of both lower limb were measured by basic behavioral scoring,cage climbing test,improved Tarlov score,MEP and SEP to determine the stability of SCI model.4.7 days after SCI of cynomolgus monkey,activated Schwann cells were injected in the injured site,each point of micro-injection 10 ul,a total of 30 ul,cell concentration: 2-3 × 105 / ul,purity> 95%,SEP,MEP and modified Tarlov scoring were used to detect the function of both lower extremities.Histological changes were observed by HE staining and immunohistochemical staining.?Results?1.After two rounds of differential digestion.The purity of SCs was(96.1 ±1.68)% in low concentration type ? collagenase digestion group and(80.1 ± 4.77)%in the control group.There was significant difference between the two groups,P<0.01.2.IL-11 can significantly increase the expression level of peripheral nerve myeli22 in SCs,while the expression level of myelin basic protein and myelin protein zero in SCs has no significant effect.The physiological effect can be inhibited by JAK-specific inhibitor AG490.The levels of pJAK2(Y1007 + Y1008)and pSTAT3(Y705)were significantly higher in SCs cells treated with IL-11 than in the control group and accompanied by the upregulation of gp130 expression in SCs.3.In the SCI group,the latent period of SEP and MEP of the lower extremities of the cynomolgus monkeys were decreased and the amplitude was decreased.The behavioral score of the SCI group was significantly lower than that of the sham operation group.4.On the Sevrnth day after SCI,SCs was injected into the injured site at a dose of 30 ?l,the total cell concentration was 2-3 × 105/ ul,and the HE staining showed obvious difference.The growth of axons in NF200 was greater than that in the injury group(p<0.05).The behavioral score of the lower limbs was significantly higher than that of the injured group(p<0.05).?Conclusion?1.The autogenous source of SCs were determined,a large number of high purity SCs was obtained using a small number of nerve tissue in a short time.And the culturing method does not use forskolin and other substances to promote mitotic agents and cytarabine and other fibroblasts to inhibit the growth of fibroblasts,which is in line with the clinical application requirements for autologous Schwann cells transplantation.2.IL-11 can promote the expression of peripheral nerve myelin 22 in SCs by regulating gp130 / JAK2 / STAT3 signaling pathway,which can activate SCs better in vitro.3.As a quantitative,objective spinal cord electrophysiological detection technology with good accuracy and high sensitivity.MEP and SEP can be used as functional evaluation basis for non-human primate animal.As a classical evaluation method,Behavioral scoring is widely adopted for its convenience and practical advantages.In this study,spinal cord hemisection injury was combined with electrophysiological and behavioral scores to ensure the consistency and accuracy of modeling.4.On the 7th day after SCI,activated Schwann cells were injected into the lesion site at a dose of 10?L,totaling 30?l,and the cell concentration was 2-3 × 105/?l.The transplantation route and the transplant dose provide guideline for the clinical treatment of SCI.