中文摘要
胶质瘢痕是脊髓损伤后修复的关键障碍，硫酸软骨素蛋白多糖（CSPGs）是其主要抑制成份，由糖胺多糖（GAG）与不同核心蛋白结合而成。CSPGs及其降解产物可通过多种途径抑制轴突再生，故抑制其形成更为重要，但目前尚缺乏有效措施。木糖转移酶-1（XT-1）是合成GAG的关键酶，抑制XT-1表达能够阻止CSPGs合成。鉴于腺相关病毒介导的RNAi具有长期、特异、高效沉默靶基因的优点，本项目通过构建特异沉默XT-1基因的shRNA的重组腺相关病毒载体； 经体外基因转染方法，用重组病毒转染星形胶质细胞（AS），检测其沉默效率及对AS增殖、活化等功能的影响，筛选出具有特异性抑制效率的RNAi病毒载体；用转染的AS与雪旺细胞、神经元共培养，探讨其促进轴突再生机制； 并通过将重组病毒注入大鼠脊髓损伤处，检测其在体内对CSPGs合成抑制效率，并评估其在脊髓修复中的作用，在一定程度上达到治疗脊髓损伤作用，恢复脊髓后肢运动功能，本课题以期为脊髓损伤治疗提供新的治疗策略，为临床应用提供理论依据。
英文摘要
The glial scar is one of the critical disturbance in spinal cord injury and its repairs, and the sulfuric acid chondroitin protein polysaccharide (CSPGs) is its mainly inhibition, which is made up of glycosaminoglycan (GAG) with the different core protein. CSPGs and its degradation product may suppress the axon regeneration by many kinds of ways. Therefore, it might be more important to inhibit CSPGs formation ,but at present, the effective action is still lacked. Xylose transferase - 1 (XT-1) is the key enzyme to synthesize GAG, inhibiting the XT-1 expression wight be able to prevent the CSPGs synthesis. In view of the fact of long-term, special and high effective silence target gene of adeno-associated virus(AVV) mediated RNAi, this project has successfully been construction the reorganization adeno-associated virus carrier of silences XT-1 gene shRNA specially, by the method of gene transfection in vitro, transfected the astrocyte with the reorganization virus, and examined its silence efficiency and asterocytes multiplies and activation influence, and screening the specificity RNAi virus carrier . and applying the transfected AS co-cultured with Schwann cells and neurons to approach the promotion axon regeneration mechanism. Then the reorganization virus were injected into the site of damaged spinal cord
结题摘要
胶质瘢痕是脊髓损伤后修复的关键障碍，硫酸软骨素蛋白多糖（CSPGs）是其主要抑制成份，由糖胺多糖（GAG）与不同核心蛋白结合而成。CSPGs及其降解产物可通过多种途径抑制轴突再生，故抑制其形成更为重要，但目前尚缺乏有效措施。木糖转移酶-1（XT-1）是合成GAG的关键酶，抑制XT-1表达能够阻止CSPGs合成。鉴于腺相关病毒介导的RNAi具有长期、特异、高效沉默靶基因的优点，本项目通过构建特异沉默XT-1基因的shRNA的重组腺相关病毒载体； 经体外基因转染方法，用重组病毒转染星形胶质细胞（AS），检测其沉默效率及对AS增殖、活化等功能的影响，筛选出具有特异性抑制效率的RNAi病毒载体；用转染的AS与雪旺细胞、神经元共培养，探讨其促进轴突再生机制； 并通过将重组病毒注入大鼠脊髓损伤处，检测其在体内对CSPGs合成抑制效率，并评估其在脊髓修复中的作用，在一定程度上达到治疗脊髓损伤作用，恢复脊髓后肢运动功能，本课题以期为脊髓损伤治疗提供新的治疗策略，为临床应用提供理论依据。