siRNA沉默波形蛋白表达抑制大鼠急性脊髓损伤胶质瘢痕的形成

Vimentin silenced by small interfering RNA inhibits glial scar formation in a rat model of acute spinal cord injury

背景:脊髓损伤后胶质瘢痕形成物理屏障抑制轴突跨越损伤部位,被认为是神经修复过程中的不利因素。目的:研究siRNA干扰波形蛋白表达对反应性星形胶质细胞的影响,探讨其在脊髓损伤后胶质瘢痕形成中的意义。方法:①选取生长良好的第3代星形胶质细胞,分为未转染组、siRNA-NC组、siRNA-Vimentin组,利用siRNA干扰技术沉默星形胶质细胞中波形蛋白表达,转染24 h进行波形蛋白免疫荧光染色,采用划痕法观察细胞迁移及增殖情况。②SD大鼠随机分为3组:正常组10只,只打开椎板,不损伤脊髓;模型组20只,制备脊髓损伤模型,脊髓内直接注射盐水和空载质粒;治疗组20只,制备脊髓损伤模型,脊髓内直接注射siRNA干扰24 h波形蛋白质粒,分别于术后1 d和4,8周观察脊髓的连续性、粗细及瘢痕与周围组织粘连情况。结果与结论:①siRNA-Vimentin组免疫荧光染色积分吸光度值低于未转染组、siRNA-NC组(P<0.05),siRNA-Vimentin组的划痕宽度大于未转染组、siRNA-NC组;②术后4,8周,治疗组损伤部位脊髓组织结构改善,胶质瘢痕形成范围小于模型组;③结果表明,波形蛋白对反应性星形胶质细胞增殖及迁移能力有显著的抑制作用,并可以抑制脊髓损伤后胶质瘢痕的形成,为脊髓损伤后轴突再生及神经功能恢复提供良好的微环境。

BACKGROUND:A barrier with formation of glial scar after spinal cord injury is considered to be harmful for nerve repair.OBJECTIVE:To explore the influence of small interfering RNA(siRNA)-mediated inhibiting Vimentin on reactive astrocytes and on the formation of glial scar after central nervous system injury.METHODS:Third-generation astrocytes that grew well were selected and randomized into untransfected group,siRNA-NC group,and siRNA-Vimentin group.We used siRNA interference technology to silence vimentin expression in astrocytes,and Vimentin immunofluorescence staining was conducted at 24 hours after transfection.Cell migration and proliferation were observed through a cell scratch test.Sprague-Dawley rats were randomized into a control group(n=10),in which only the lamina was opened,without damage to the spinal cord;a model group(n=20),in which a spinal cord injury model was prepared,followed by direct injection of saline and empty plasmids into the spinal cord;and a treatment group(n=20),in which the spinal cord injury model was prepared followed by direct injection of Vimentin plasmids intervened with siRNA for 24 hours.Spinal cord continuity and thickness as well as scar adhesion with surrounding tissues were observed at 1 day,4,and 8 weeks after surgery.RESULTS AND CONCLUSION:Integral asorbance value of Vimentin by fluorescence immunoassay was lower in the siRNA-Vimentin group than the untransfected and siRNA-NC groups(P<0.05).The scratched width was larger in the siRNA-Vimentin group than the untransfected and siRNA-NC groups.The spinal cord tissue structure at the injured site was improved in the treatment group,and the range of glial scar formation was smaller in the treatment group than the model group at 4 and 8 weeks after surgery.To conclude,Vimentin has a significant inhibitory effect on the proliferation and migration of reactive astrocytes and inhibits the formation of glial scars after spinal cord injury,providing a favorable microenvironment for axonal regeneration and nerve function recovery after spinal cord injury.