新型胶原蛋白纳米纤维支架复合GDNF修饰的间充质干细胞修复脊髓损伤的实验研究

Spinal cord injury is a serious central nervous system traumatic disease. Aplastic neurons and axons, the lack of neurotrophic factor and glial scar formation are main influencing factors for spinal cord injury repairment. Our previous study demonstrated that aligned oriented collagen nanofibers could promote neural stem cells proliferation and expansion, and improve glial scar. Increasing evidence suggested that the therapeuticpotential of mesenchymal stem cell （MSC） could be applied to neural injury repairment. Glial cell line-derived neurotrophic factor (GDNF), a multifunctional neurotrophic factor known to affect neuron proliferation and survival, axon regeneration. Thus, in the present study, we will prepare the GDNF gene modified BMSC, and aligned oriented collagen nanofibers, then determine the synergetic therepeautic effect on spinal cord injury. To test the proposal, we will determine the effect of the secreted GDNF on BMSC differention, survival and proliferation, and the effect of GDNF-modified BMSC on neuron proliferation and survival in vitro. Meanwhile, we will determine the effect of aligned oriented collagen nanofibers on GDNF secretion and GDNF-modified BMSC function and morphology.Using rat model of spinal cord injury, we will invesigate the synergetic therapeutic effect of transplantations of GDNF- modified BMSC combined with collagen nanofibers via determination of motor function and graft pathologic histology.Furthermore, we will explore the underlying mechanisms of the combined treatment strategy by investigating MSC engraft, differentiation, survival,neuron and axon regeneratio, cell apoptosis and expression of neurotrophic factors. This study may provide a new strategy for treatment of spinal cord injury, even other tissue neural injury.

脊髓损伤是一种严重的中枢神经系统创伤性疾病。而影响脊髓损伤修复最主要的因素是神经元及轴突的再生障碍，神经营养因子缺乏以及胶质疤痕形成等。我们以往工作发现新型胶原蛋白纳米纤维可以有效的改善胶质疤痕生成，促进神经干细胞的增殖。本课题拟在前期基础上，制备胶质细胞源性神经营养因子（GDNF）转染的骨髓间充质干细胞（BMSC），协同发挥干细胞神经分化能力和GDNF的促神经细胞增殖生长作用；同时将胶原蛋白纳米纤维作为支架，一方面可以抑制胶质疤生成，另一方面为神经营养因子和干细胞提供更多接触机会，进一步增强它们的修复功能。为此，我们将从体外研究GDNF对BMSC的形态及分化能力的影响；制备新型定向型胶原蛋白纳米纤维，研究其对BMSC的影响；建立大鼠脊髓损伤模型，研究该复合移植物对大鼠脊髓损伤的治疗效果，并探讨修复机制。本研究将为干细胞移植治疗提供一种新的尝试，也为脊髓损伤的治疗提供新的思路。

脊髓损伤（SCI）因其高致残率严重威胁人的生存质量，损伤后的修复是世界性的重点和难题。而影响脊髓损伤修复最主要的因素是神经元及轴突的再生障碍，神经营养因子缺乏以及胶质疤痕形成等。本研究我们从体外研究GDNF 对BMSCs 的形态及分化能力的影响；建立大鼠脊髓损伤模型，研究对大鼠脊髓损伤的治疗效果。结果发现：我们成功构建携带绿色荧光蛋白标记的GDNF 慢病毒载体，并成功的转染BMSCs。我们发现BMSCs表达高水平GDNF mRNA但表达低水平GDNF蛋白。生物信息学分析表明,mir-383通过结合在GDNF mRNA的3`端的非编码区，从而抑制GDNF的蛋白质翻译。与单纯应用BMSCs相比较，用携带了反义miRNA-383的腺相关病毒复合体（AAV-as-miR-383）转染的BMSCs进一步提高了SCI后裸鼠的完整组织比例，减少了腔体积容量，增强运动活性的恢复。通过本课题的研究发现，单纯提高GDNF mRNA水平并非意味GDNF蛋白高表达，而通过抑制miR-383，可增加GDNF的蛋白表达水平，从而增加人骨髓间充质干细胞治疗脊髓损伤的潜能。