siRNA干扰EphB2抑制胶质疤痕形成及组织工程修复脊髓损伤

One of the obstacles for neural regeneration following spinal cord injury (SCI) is the rapid formation of dense glial scar, through which the slow outgrowing axons can hardly pass. Recent studies indicated that the overcoming of glial scar is out of the reach of existing tissue engineering, which has emerged as a potentially promising repair for many tissues and organs. Although some approaches have been found to facilitate glial scar removal in varying degrees, complete inhibition of the glial scar formation seldom occurs after this sort of afterwards treatment. Recent studies have shown that one of the early pathophysiological events after SCI is up-regulated expression of EphB2 receptor in fibroblasts as well as epherin-B2 ligand in astrocytes, arousing phosphorylation of EphB2 and epherin-B2 by binding them each other. Therefore, EphB2 or its ligand may represent a potential target for glial scar. We hypothesized that inhibition of the up-regulation of EphB2 or its ligand could prevent glial scar formation from the very beginning. Based on this, we will seek to determine, in the present project, the possible inhibition of glial scar formation by down-regulation of EphB2 with RNAi treatment. Furthermore, we will observe whether improved repair outcome can be achieved by incorporating EphB2 RNAi with regionally tissue-engineered spinal cord graft, which has been successfully fabricated in our lab during a previous NSFC project. Relevant experimental techniques and methodology of molecular and cell biology and neurobiology will be utilized to test our hypothesis. New pharmaceutical target and approach may be anticipated to use RNAi for the treatment of SCI, providing a way to promisingly overcome the glial scar barrier for axonal regeneration after the injury of the central nervous system.

脊髓损伤难以修复的原因之一是快速出现的致密胶质疤痕成为生长相对缓慢的轴突难以逾越的障碍，且已有的组织工程修复技术也难以解决；目前虽有专门消除胶质疤痕的方法，但都属事后补救的措施，不能全面阻止它的抑制作用。研究已发现，脊髓损伤最初变化之一是成纤维细胞EphB2受体和星形胶质细胞ehprin-B2配体表达上调并借此结合而磷酸化，然后两细胞协同形成胶质疤痕。因此若能抑制这一受体或配体的表达上调，可能是从源头阻止胶质疤痕形成的为数不多且有效的靶点。本课题拟在脊髓损伤之初，用RNAi技术使EphB2表达不上调，两种细胞无法借此结合形成胶质疤痕；同时结合前一个项目的"分区式组织工程脊髓"，争取进一步提高修复损伤脊髓的效果；实验将以相应的分子生物学、细胞生物学和神经生物学技术及体内外实验相结合方法来验证。此研究成功将可能为RNAi治疗脊髓损伤提供新手段，也可在消除脑损伤后胶质疤痕所带来的负面影响时被借鉴

当脊髓损伤（SCI）时，快速出现的胶质-纤维疤痕致密组织将成为生长相对缓慢的再生轴突难以逾越的障碍，从而成为SCI难以修复的主要原因之一，即使是已有的组织工程修复技术也难以解决这一临床医学问题。虽然目前针对胶质疤痕消除的方法有多种，但基本都属于事后补救的消除疤痕措施，并不能从源头阻止它的形成及其解除其的抑制作用。从已有的研究中发现，SCI最初的分子变化事件之一，有成纤维细胞的EphB2受体和星形胶质细胞的ehprin-B2配体表达上调，并借此相互结合而引发磷酸化，然后两种细胞协同作用，分泌细胞外基质，最终形成胶质-纤维疤痕。因此若能抑制这一受体或配体的表达上调，可能是从源头阻止胶质-纤维疤痕形成的为数不多且有效的靶点。本课题拟在SCI之初，用RNAi技术使EphB2表达不能急剧上调，从而阻止两种细胞不能完全借此亲和对结合，而适度控制胶质-纤维疤痕的形成程度；同时结合前一个国家自然科学基金项目的“分区式组织工程脊髓”，促进再生轴突的生长能力，争取进一步提高修复损伤脊髓的效果。我们分别采用了针对EphB2和ehprin-B2的有效干扰基因序列，在使用TGF-β1作用或划痕作用的胶质-纤维疤痕体外模型上，能使所形成的胶质-纤维疤痕细胞团簇样结构数量变少，直径也明显减小；ELISA结果显示RNAi后，aggrecan和versican的表达量显著下降；用微流控芯片技术发现，VSC4.1细胞的细长轴突，在有RNAi的情况下，生长长度明显大于无干扰的对照组。而在体内的SCI模型上，施加RNAi，EphB2表达量明显降低、硫酸软骨素蛋白聚糖等表达也显著减少；同时增加NT3作用，电生理及行为学实验结果也显示大鼠的后肢功能恢复良好；也观察SCI并应用RNAi后，血脑屏障的通透性及炎症细胞和因子的渗透并未明显增加。另外，增加了在“分区式组织工程脊髓对大鼠SCI后神经组织的细胞凋亡的影响”、“应用RNA-Seq技术研究SCI急性/亚急性期的基因表达差异”和“SCI后miR-34s、miR-17的变化趋势及其作用”三方面的研究。此方面研究将为RNAi治疗SCI提供新手段，也可在消除脑损伤后胶质疤痕所带来的负面影响时被借鉴。