| Objective: To observe the protective effect of N-acetylglucosaminyltransferase V(Mgat5)lentiviral vector on the expression of β-1,6-Glc NAc-branched N-glycans in local tissue after spinal cord injury(SCI)in rats,and explore the possible mechanism of protective effect.Methods:(1)The lentivirus vector of rat Mgat5 was constructed and titers of the lentiviral vector was estimated.(2)Separated and identificated the purity of primary rat dorsal root ganglion(DRG)cells.(3)Treated DRG cells with different multiplicity of infection(MOI)for 24 h,48 h and 72 h,the transfection efficiency was observed by inverted fluorescence microscope to determine the best transfection MOI.(4)Days 1,3,5 and 7 after DRG cells were transfected with the best MOI,the cytotoxicity of Mgat5 lentivirus vector to neurons was detected by CCK8.(5)The optimal MOI was transfected into DRG cells for day 7,and the expression levels of β-1,6-Glc NAc-branched N-glycans in DRG cells were detected by PHA-L binding assay.(6)Take 3 female SD rats(200-220 g),constructed spinal cord injury model by vascular clamp,and a micro syringe was use to inject 11 μL negative control Lentivirus at two different locations of the spinal cord(1.5 mm from the injury center).Lentivirus was injected at a depth of about 1 mm.Spinal cord tissues were taken for frozen sections at days 3,7,and 14,and the effect of lentivirus infection in vivo was observed under a fluorescent microscope.Another 120 rats were randomly divided into 5 groups: simple injury model group(laminectomy and SCI,abbreviated as SCI group),experimental group(laminectomy and SCI and Lv-Mgat5,abbreviated as Lv-Mgat5 group),no-load lentivirus group(laminectomy and SCI and Lv,abbreviated as Lv group),methylprednisolone group(laminectomy and SCI and methylprednisolone group,abbreviated as MP group),sham operation group(only laminectomy,abbreviated as Sham).After successful modeling,the motor function was detected by BBB score and inclined plate function score in 3th,7th and 14 th day.After observing the changes of neurological function,the injured spinal cord tissue was taken to detect the expression level of β-1,6-Glc NAc-branched N-glycans by PHA-L binding assay,and the expression of Mgat5 and GAP-43 in each group were detected by western-blot method.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of inflammatory cytokines IL-1 β,IL-10 and TNF α in each group.Results:(1)The lentiviral vector expressing Mgat5 was successfully constructed.(2)The primary DRG cells of adult rats were successfully cultured and the purity was 90%.(3)Mgat5 lentiviral vector successfully infected rat DRG cells,and MOI=10 was determined as the best transfection dose.(4)The results showed that 1th,3th,5th and 7th day after transfection,Mgat5 lentivirus had no significant effect on neuronal activity.(5)PHA-L binding assay showed that lentivirus carried Mgat5 could significantly increase the level of β-1,6-Glc NAc-branched N-glycans in DRG cells.(6)The spinal cord tissue of rats had strong fluorescent expression at various time points after transfection with lentivirus,indicating that the lentivirus can be continuously and stably expressed in the injured area of spinal cord.(7)The results of BBB score showed that the hind limbs of rats were completely paralyzed after SCI,and the BBB score was stable at 0,indicating that the animal model was successfully established and the injury was consistent.BBB score and inclined plate test showed that compared with the score of SCI only group,MP group and Lv-Mgat5 group was significantly higher,but there was no significant difference between the Lv group and the SCI model group.(8)The results of the PHA-L binding test of spinal cord tissue showed that compared with SCI group,the expression of β-1,6-Glc NAc-branched N-glycans in the Lv-Mgat5 group was significantly increased on 3th,7th and 14 th day,while MP group was increased on 3th day,and there was no significant difference in other groups at each time point.(9)Western blot results showed that compared with the SCI group,the expression of Mgat5 protein in Lv-Mgat5 group increased significantly on the 3th,7th and 14 th day after inury,and the expression of Mgat5 protein in MP group increased only on the 3th day.Compared with the SCI group,the expression of Mgat5 protein in other groups had no significant difference at each time point.Compared with SCI group,the expression of GAP-43 protein had no significant change in all groups on the 3th after injury,but the expression of GAP-43 protein in Lv-Mgat5 group was significantly higher than that in SCI group on the 7th,14 th day after injury,while the expression of GAP-43 protein in MP group only increased on the day 7 after injury.(10)The results of ELISA showed that the expression levels of IL-1 β and TNF α in the MP group and the Lv-Mgat5 group were significantly lower than those in SCI group,while the expression level of IL-10 in the MP group increased significantly at all three time points,while in Lv-Mgat5 group,the expression level of IL-10 began to increase on the 7th day after injury and lasted to 14 th day,and there were significant differences between the two groups and SCI group.Conclusion: Mgat5 lentiviral vector was successfully constructed in this study.The lentivirus vector can be stably infected in vivo and in vitro,and has no significant effect on cell growth.Lentivirus infection can increase the expression level of β-1,6-Glc NAc-branched N-glycans in cells and spinal cord tissue.Local injection of Mgat5 lentivirus vector can promote axonal regeneration of injured spinal cord and accelerate the recovery of motor function of hindlimb in rats.This study proves that lentiviral vector-mediated Mgat5 may be one of the effective methods for SCI therapy,and its possible mechanism is that the recombinant lentiviral vector can stably express Mgat5 in spinal cord nerve tissue,thereby increasing the level of β-1,6-Glc NAc-branched N-glycans in spinal cord tissue.By down-regulating the pro-inflammatory cytokines TNF-α,IL-1-β,and up-regulating the expression of anti-inflammatory factor IL-10,reducing the inflammatory response in the injured spinal cord and promoting the expression of GAP-43 in the injured spinal cord tissue,this ultimately promotes the regeneration of nerve axons in the spinal cord injury area. |
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