An Experimental Study On The Effect Of Interleukin-10 In BMSCs On The Repair Of Spinal Cord Injury

An Experimental Study on the Effect of Interleukin-10 in BMSCs on the Repair of Spinal Cord InjuryBackgroundSpinal cord injury(SCI) contributes to the disorders of long descending motor and ascending sensory pathways and induces complete or partial functional loss of motor and sensory nerves.SCI was classified into two pathological stages:primary and secondary injury.The former was companied by hemorrhage,edema,necrosis and acute inflammation with the release of inflammatory cytokines,toxic free radicals,and protease. While delayed cell death,demyelination,chronic inflammation,and reactive gliosis, occurring over days to weeks,are showed in the secondary injury.The traditional treatment(including surgery,drugs,physical recovery,etc.) works but couldn't stop the progress of this kind of injury.As the development of techniques and theory of stem cell,using bone marrowmesenchymal stem cells(BMSCs) to treat SCI showed a promising prospect.The previous studies found that the function of nerves damaged by SCI improved obviously after BMSCs transplantation.However,the mechanism involved in this process remains unclear.Studies revealed that BMSCs could secret certain cytokine which could inhibit inflammation and thus relieve or stop the secondary injury.BMSCs itself could secret IL-10,which is an important inhibitor of regulating immune response and an anti-inflammatory cytokine.So far,the effect of IL-10 in BMSCs on the repair of SCI was rarely reported.The aim of this study was to investigate the changes of IL-10 in SCI treated by BMSCs;AdCMV-mouse IL-10 was transferred into BMSCs which then were transplanted into injured spinal cord of mouse.The therapeutic effect of trans-IL-10 gene BMSCs on the recovery of neural function after spinal cord injury was also investigated.This experiment provides a potential clue for the gene-therapy for SCI.Objective1.BMSCs were separated and purred by magnetic beads assay from marrow of the wild C57BL/6 mouse and the C57BL/6 mouse whose IL-10 was knock-out.2.To investigate the expression and changes of IL-10 in injured spinal cord after treated by BMSCs. 3.AdCMV-mouse IL-10 was constructed and transferred into BMSCs in vivo to observe its effect on the proliferation of BMSCs.BMSCs with IL-10 overexpression were collected and acted as a promising target for gene therapy.4.To investigate the role of BMSCs with IL-10 overexpression on the function changes of SCI.Methods1.Magnetic beads assay was used to separate BMSCs from marrow of the wild mouse and the mouse whose IL-10 was knock-out.The BMSCs were cultured and generated in vivo.Its characteristics were observed.The flow cytometry was used to detect the biomarkers of stem cells and then to identify their characteristics.2.The mouse with SCI was classified into three groups:group A(treated with wild BMSCs),group B(treated with IL-10(-/-) BMSCs) and control group(treated with PBS).RT-PCR was used to detect the levels of IL-10 and TNF-a mRNA of SCI.BBB score and HE staining was taken to observe the status of injured spinal cords.The tissue section of spinal cords were separated and inspected by fluorescence microscope.3.IL-10 cDNA was got from mouse through RT-PCR.Then AdCMV-mouse IL-10 was constructed according to AdEasyTM-1 Vector System.AdCMV-mouse IL-10 was transferred into BMSCs in vivo and then IL-10 was detected at the DNA and protein level.The survival curves were made to observe the effect of IL-10 on BMSCs.4.IL-10-BMSCs was transplanted into the injured spinal cords.RT-PCR was again used to detect the levels of IL-10 and TNF-a mRNA of SCI.BBB score and HE staining was taken to observe the status of injured spinal cords.Results1.The BMSCs were fibroblast-like and grow fast in vivo in type of whirlpool.The biomarkers of CD29 and CD44 were detected on 99%BMSCs.2.Expression of IL-10 in group A was significantly higher than that in group B and control group(P＜0.01) at day 14,21,and 28 after treatment.No significant difference was found between group B and control group(P＞0.05).The level of TNF-a mRNA was significantly lower in group A than that in group B and control group(P＜0.0 5 and P＜0.01).TNF-a mRNA level was higher in group B than that in control group.Similarly,the recovery status in group A was better in group A than in group B and control group.The labeled -cells was migrated as far as 2-4mm along the rostrocaudal axis in the spinal cords at 28 days post-transplantiations.So these results confirmed the ability of the BMSCs cells to survive for long intervals(28days)in vivo and to migrate in the injured spinal cords.3.We successfully constructed AdCMV-mouse IL-10 and transferred it into BMSCs. BMSCs can secret IL-10 and the amount of IL-10 was associated with the amount of AdCMV-mouse IL-10 transferred.However,the proliferation of BMSCs was not affected.4.After transplantation,the level of IL-10 and TNF-a mRNA changed greatly between test group and control group.IL-10 mRNA expression was significantly higher in test group than that in control group(P＜0.01).The level of TNF-a mRNA,in contrast, was significantly lower in test group than that in control group(P＜0.01).The recovery status in test group was better than in control group.Conclusions1.Highly pure BMSCs from marrow of the wild mouse and the mouse whose IL-10 was knock-out have a multi-differentiate potential.The biomarkers on cell surface were positive for stem cells.2.BMSCs could secret IL-10 and then promote the recovery of injured spinal cords.This result highlights the mechanism of BMSCs on the repair of SCI and provides an important theory for gene therapy.3.The successfully constructed AdCMV-mouse IL-10 was proved to be an ideal tool for the study of the role of IL-10 in BMSCs.IL-10 level was increased after transfect.The model could be used to study the effect of IL-10 on the repair of SCI.4.BMSCs with IL-10 overexpression have a better effect than that with IL-10 low-expression or lost-expression.It would be a good and potential method to promote recovery of SCI through enhancing the expression of IL-10 in BMSCs.