| Objective:To observe the vascular remodeling and inflammatory response after overexpression of microRNA-126 bone marrow mesenchymal stem cells(BMSC)in spinal cord of(SCI)rats with spinal cord injury,and to evaluate the effect of overexpression of microRNA-126 BMSC on the recovery of neurological function after spinal cord injury.Methods:1.The lentivirus vector system overexpressing miR-126 was constructed and identified by whole bone marrow adherent culture of bone marrow mesenchymal stem cell(BMSC).The lentivirus stably overexpressing miR-126 was screened and transfected into BMSC by 293 T cell packaging.Establishment and experimental grouping of spinal cord injury model in.2.SD rats 108 rats were randomly divided into three groups: simple spinal cord injury group(control group,n = 36),spinal cord injury blank vector BMSC transplantation group(experimental group Ⅰ,n = 36)and spinal cord injury overexpression miR-126 BMSC transplantation group(experimental Ⅱ group,n = 36).3.The rats in each group were perfused at 3 days,7 days and 28 days after spinal cord injury.HE staining was used to observe the morphology and cavity size of the injured spinal cord,and Nissan body staining was used to observe the regeneration of Nissan body.4.The rats in each group were perfused at 1 day,3 days and 7 days after spinal cord injury for double immunofluorescence staining of endothelial cell specific antibody(RECA-1)and microglia marker(CD68).The axons(N52)and astrocytes(GFAP)were stained by double immunofluorescence at 1 week and 4 weeks after spinal cord injury.Quantitative analysis of the positive number of immunofluorescence staining indicators,analysis of vascular remodeling,inflammatory reaction and nerve growth.5.At 12 hours,1 day,3 days and 7 days after operation,the contents of IL-1 β,IL-6 and TNF-α in spinal cord tissue were measured by ELISA,and the inflammatory reaction was analyzed.6.The neurological function was evaluated by BBB score on the 1st,3rd,7th,14 th and 28 th day after operation.Results:1.Bone marrow mesenchymal stem cells(BMSC),)were successfully cultured and overexpressed miR-126 lentivirus vector system was successfully constructed.the stable expression of BMSC was transfected and screened.2.HE staining and Nissl body staining: HE staining showed that BMSC transplantation group(experimental group Ⅰ)and overexpression MicroRNA-126 BMSC transplantation group(experimental Ⅱ group)injured spinal cord cavity were reduced to varying degrees.The cavity area decreased more obviously in the experimental Ⅱ group.Nissl staining showed that more Nissl bodies were found in the experimental Ⅱ group,and the volume was closer to the normal Nissl body volume than that in the experimental group Ⅰ.3.Double immunofluorescence staining of RECA-1 and CD68: a large number of CD68 positive cells were observed in the control group 3 days after injury.the number of inflammatory cells in group Ⅰ and II was significantly lower than that in the control group(P < 0.01),and the number of inflammatory cells in the experimental Ⅱ group was the least.The RECA-1 positive vessel density was the highest in the experimental Ⅱ group,but lower in the experimental Ⅰ group and the control group(P < 0.05 and P < 0.01,respectively).Seven days after injury,there were lower density of CD68 positive cells in experimental Ⅱ group than in experimental group Ⅰ and control group(P < 0.05 and P < 0.01,respectively),and the number of CD68 positive cells in experimental group Ⅰ was lower than that in control group(P < 0.05).The vascular density in the experimental Ⅱ group was higher than that in the experimental Ⅰ group and the control group(P < 0.01).4.Double immunofluorescence staining of N52 and GFAP: one week after operation,GFAP astrocytes gathered in the injured spinal cord area,and the number of GFAP positive cells in the experimental Ⅱ group was lower than that in the experimental Ⅰ group and the control group(P < 0.01).The number of N52 positive nerve fibers in the experimental Ⅱ group was significantly higher than that in the experimental Ⅰ group and the control group,and the number of N52 positive nerve fibers in the experimental Ⅰ group was significantly higher than that in the control group(P < 0.01).At 4 weeks after operation,the number of glial scar formed by astrocyte proliferation in GFAP group was significantly higher than that in the other two groups(P < 0.01),and the number of scar tissue in experimental Ⅱ group was smaller than that in experimental group Ⅰ(P < 0.05).There were the most N52 positive axons in the experimental Ⅱ group,which were significantly higher than those in the experimental Ⅰ group and the control group(P < 0.01 and P < 0.05,respectively),and the number of positive axons in the experimental Ⅰ group was significantly higher than that in the control group(P < 0.01).5.The contents of IL-1 β,IL-6 and TNF-α detected by ELISA: the contents of IL-1 β,IL-6 and TNF-α in the injured spinal cord of the experimental Ⅱ group were significantly lower than those of the control group at 12 hours,1,3 and 7 days after injury(P < 0.01).12 hours after injury,the content of TNF-α in experimental Ⅱ group was significantly different from that in experimental group Ⅰ(P < 0.05).One day after injury,the contents of IL-6 and TNF-α in Ⅱ group were significantly different from those in group Ⅰ(P < 0.05).Three days after injury,the contents of IL-1 β and TNF-α in Ⅱ group were significantly different from those in group Ⅰ(P < 0.05).Seven days after injury,the contents of IL-1 β,IL-6 and TNF-α in experimental Ⅱ group were significantly different from those in experimental group Ⅰ(P < 0.01).6.BBB score: on the 3rd,7th,14 th and 28 th day after operation,the BBB score in the experimental Ⅱ group was significantly higher than that in the control group(P < 0.01),and there was a significant difference on the 1st day after operation(P < 0.05).The BBB score of experimental Ⅱ group was significantly higher than that of experimental group Ⅰ on the 14 th and 28 th day after operation(14 days P < 0.05,28 days P < 0.01).The BBB score of group Ⅰ was significantly higher than that of the control group on the 3rd,7th,14 th and 28 th day after operation(P < 0.05 on the 3rd day,P < 0.01 on the remaining day).Conclusion:The transplantation of miRNA-126-overexpressed BMSC into spinal cord injury rats can promote the regeneration of spinal cord blood vessels and the recovery of nerve function,and inhibit the secondary inflammatory reaction in the injured area.Overexpression of miRNA-126 BMSC is more effective than BMSC in the treatment of spinal cord injury. |
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