Sox2质粒诱导成纤维细胞直接重编程为神经干细胞复合多层碳纳米管支架治疗脊髓损伤的实验研究

Although axon is renewable after spinal cord transection, it couldn’t effectively across the scar interface. It becomes a principle problem restricts the treatment of spinal cord injury. Our study has confirmed that neural stem cells differentiated from induced pluripotent stem cells (iPS) combined with scaffold materials could be effectively utilized to repair spinal cord injury. However, we also found that induction of iPS had following shortcomings, low induction efficiency, time consuming, and tumorigenic potential. Protein-induced iPS may decrease its tumorigenic potential, but its efficiency was not high enough, and usually difficulty to replicate. If we can bypass the multi -potential stem cell process, direct reprogramming somatic cells into required cell could be an effective solution to this issue? In view of the above assumptions, based on our successful experience of induction iPS use plasmid, we intend to induce mouse fibroblasts directly programming into neural stem cells (iNSC) using single factor plasmid Sox2. And utilize multi-wall carbon nanotubes with supreme adhesion and neuronal induction properties. Explore iNSCs adhesion, proliferation, differentiation and other biological behavior on the scaffold. And transplant iNSC-seeded scaffolds in rats model of spinal cord injury to explore whether the tissue engineered spinal cord could promote axonal regeneration across scar interface, form functional neural connections to achieve the purpose of partial repair and reconstruction of the injured spinal cord.

脊髓横断后，轴突虽可再生但不能有效通过瘢痕界面已成为制约脊髓损伤治疗的关键问题。我们研究已证实，诱导多能干细胞（iPS）定向分化神经干细胞复合神经支架材料可有效修复脊髓损伤。但研究亦发现iPS诱导效率低、耗时长、诱导不完全具有潜在的致瘤性。蛋白诱导iPS虽可降低其潜在致瘤性，但其效率低、重复性差。如能绕过多潜能干细胞状态，直接将体细胞重编程为所需细胞可否有效解决这一问题？针对以上假设，我们在应用质粒成功诱导小鼠iPS的基础上，首次拟用Sox2单因子质粒诱导小鼠成纤维细胞直接重编程为神经干细胞（iNSC）；并应用具有良好的粘附、诱导定向生长性能的多层碳纳米管构建纳米结构支架，体外研究该支架对iNSC黏附、增殖、分化等生物学行为影响，并将iNSC复合支架植入大鼠脊髓损伤模型，检测该组织工程化脊髓能否有促进轴突的再生通过瘢痕界面，形成功能性的神经连接，达到部分修复和重建脊髓的目的。

诱导多能干细胞（iPS）定向分化为神经干细胞（NSC）复合神经支架材料可有效修复脊髓损伤。但iPS诱导效率低、耗时长、诱导不完全具有潜在的致瘤性。蛋白及小分子诱导iPS虽可降低其潜在致瘤性，但其效率低、重复性差。如能绕过多潜能干细胞状态，直接将体细胞重编程为所需细胞，可能有效解决这一问题。本课题组在过去四年中已完成了1.在应用质粒成功诱导小鼠iPS的基础上，首次拟用Sox2单因子质粒结合电转染诱导小鼠成纤维细胞重编程为神经干细胞（iNSC）及鉴定；2通过Mn离子与变构合成的PPy结合，降低毒性，合成具有良好的弹性模量、自愈合性、导电性、生物相容性的微量锰导电水凝胶，3在体外设计一种电刺激装置研究该支架对iNSC黏附、增殖、分化等生物学行为影响，4将iNSC复合支架植入大鼠脊髓损伤模型，检测该组织工程化脊髓在促进轴突的再生，修复和重建脊髓方面的作用。