具有生物活性三维纳米神经导管的构建及其修复大鼠脊髓损伤的实验研究

Spinal cord injury (SCI) remains one of the most devastating conditions among neurological diseases, which results in paralysis below the level of the injury.Astrocytes are responsible for the formation of the glial scar, which is recognized as a barrier to regeneration by inhibiting the crossing of axons through the injuried area. In this study, we aim to design and fabricate a 3-D multichannel nerve conduits loaded with bioactive factor by coaxial electrospinning for spinal cord regeneration in a rat spinal cord hemisection injury model.The nanofibrous mat with core-shell structure (inner nanofibers is silk as core, outer nanofibers is hyaluronic acid as shell) is synthesized using coaxial electrospinning.This study will also explore the molecular mechanism of how nanofibrous scaffold modulates stem cells behaviour including adhesion, proliferation, differentiation, and improvement of the axon regeneration and remyelination. The synthesized 2-D nanofibrous mat will be fabricated to 3-D multichannel nerve conduits by rolling the silk mat with metal wires and gel spinning. The multiple channel nerve bridges will be placed in hemi-transected rat spinal cord to observe its supporting effect on axon regeneration.The study will explore the process of axon remyelination and the outcome of exogenous neural stem cells and three-dimensional nerve conduits, which will provide a scientific basis for the clinical application of tissue engineered spinal cord.

脊髓损伤后局部被瘢痕组织所替代，再生的神经轴突很难越过瘢痕组织而形成有效的突触联系。本研究拟采用同轴电纺技术，体外合成与天然细胞外基质结构和功能相似、并能缓释生物活性因子的新型纳米仿生支架膜，通过体外实验观察神经干细胞在2-D（二维）膜材料上黏附、生长、增殖和分化的生物学行为，以及脊神经节细胞轴突在该纳米膜材料上延长的情况，在细胞和分子水平上阐明神经干细胞和轴突与支架材料的界面反应机理。并进一步将新型纳米仿生支架膜制作成具有引导轴突方向性再生的3-D（三维）神经导管，构建3-D组织工程化脊髓，检测其对完全横断性脊髓损伤的修复作用，同时探讨轴突再髓鞘化、外源性神经干细胞及三维纳米支架在体内的转归，为组织工程化脊髓的临床应用提供科学依据。

脊髓损伤后局部被瘢痕组织所替代，再生的神经轴突很难越过瘢痕组织，形成有效的突触联系。本研究拟用同轴电纺技术，体外合成与天然细胞外基质结构和功能相似、并能缓释生物活性因子的新型纳米仿生支架膜，并将其制作具有引导轴突方向性再生的 3-D（三维）神经导管。制备以丝素蛋白为原料的纤维定向排列及非定向排列的纳米纤维膜，与神经干细胞共培养后发现：神经干细胞的增殖行为及被BrdU标定的细胞比率提高了30%，而且在纤维定向排列的纳米纤维膜表面的神经干细胞增殖能力更强。与纳米纤维支架共培养的细胞经周期检查发现处于 G0/G1 期的细胞比率减小，处于增殖期（S期加G2/M期）的细胞比率增加。这种增殖效应可被抗β1整合素抗体和促分裂原活化蛋白激酶激酶（MEK）抑制物U0126所消除。同时神经干细胞的细胞周期蛋白基因D1和CDK2的表达增加。构建纳米组织工程化脊髓修复脊髓半切损伤，从BBB评分，组织学检查，Brdu标定细胞，体感诱发电位等方面研究修复效果，定向纳米纤维膜组织工程化脊髓对脊髓损伤的修复效果最佳。研究结果表明：纳米组织工程支架可通过β1整合素抗体和促分裂原活化蛋白激酶途径调节细胞周期的变化，构建组织工程化脊髓修复脊髓损伤可获得良好的效果。