中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (20): 3190-3195.doi: 10.12307/2022.619

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

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

李剑锋1，张淑莲2，闫金玉2，李佳艺1，金  朝1，邓  琦2   

  1. 1北京市第二康复医院，北京市 100038；2内蒙古医科大学第二附属医院，内蒙古自治区呼和浩特市 010030
  • 收稿日期:2021-09-08 接受日期:2021-11-04 出版日期:2022-07-18 发布日期:2022-01-20
  • 通讯作者: 邓琦，副主任医师，内蒙古医科大学第二附属医院，内蒙古自治区呼和浩特市 010030
  • 作者简介:李剑锋，男，1980年生，内蒙古自治区呼和浩特市人，2015年天津医科大学毕业，博士，副主任医师，主要从事脊髓损伤基础及临床研究。
  • 基金资助:
    国家自然科学基金支持项目(81560212)，项目负责人：李剑锋；内蒙古自然科学基金支持项目(2015MS0898)，项目负责人：李剑锋

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

Li Jianfeng1, Zhang Shulian2, Yan Jinyu2, Li jiayi1, Jin Zhao1, Deng Qi2   

  1. 1Second Rehabilitation Hospital of Beijing, Beijing 100038, China; 2Rehabilitation Department, the Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010030, Inner Mongolia Autonomous Region, China
  • Received:2021-09-08 Accepted:2021-11-04 Online:2022-07-18 Published:2022-01-20
  • Contact: Deng Qi, Associate chief physician, Rehabilitation Department, the Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010030, Inner Mongolia Autonomous Region, China
  • About author:Li Jianfeng, MD, Associate chief physician, Second Rehabilitation Hospital of Beijing, Beijing 100038, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81560212 (to LJF); the Natural Science Foundation of Inner Mongolia Autonomous Region, No. 2015MS0898 (to LJF)

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摘要：

文题释义：
胶质瘢痕：是脊髓损伤慢性阶段反应性星形胶质细胞增殖所引起的，是脊髓损伤后机体自我保护性反应。胶质瘢痕主要包括细胞外基质、纤连蛋白、层粘连蛋白和胶原等。胶质瘢痕和囊腔的形成是阻碍神经再生的主要屏障。
急性脊髓损伤：通常是由于突发的脊柱椎体骨折、脱位的同时，造成脊髓的压迫伤，其病理机制主要包括早期阶段机械性损伤和次生损害的双重作用机制，多造成损伤水平以下感觉、运动及括约肌功能障碍等。

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。

https://orcid.org/0000-0002-5711-4346 (李剑锋) 

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 小干扰RNA, 波形蛋白, 星形胶质细胞, 胶质瘢痕, 脊髓损伤

Abstract: 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.

Key words: small interfering RNA, Vimentin, reactive astrocyte, glial scar, spinal cord injury

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