控释VEGF/NT-3脊髓脱细胞支架在SCI模型中的血管化及神经再生研究

Local vascular reconstruction is in close relationship with secondary injury after spinal cord injury (SCI). However, the key role of angiogenesis in SCI was neglected in previous studies. The relationship between angiogenesis and nerve regeneration after microcirculatory disturbance in SCI remains unclear. The present study is dedicate to preparing scaffolds of acellular spinal cord (ASCS) with controlled release of vascular endothelial growth factor (VEGF) and neurotrophin-3 (NT-3) on the basis of our previous research. We are looking forward to exploring the appropriate time in treatment of a spinal cord hemisection model with continuous release of VEGF and NT-3 in ASCS with green fluorescent protein (GFP) transfected embryonic neural stem cells (ENSC) derived from spinal cord of Sprague-Dawley (SD) rats. The extent of angiogenesis will be appraised by micro-CT with three-dimensional reconstruction of spinal cord vascularity, with the assistance of data analysis and calculation of vascular density. Cell differentiation of ENSC and regeneration of axon and myelin sheath can be observed by immunofluorescence staining. Meanwhile, we want to investigate the morphology of capillaries and myelin sheathes with transmission electron microscope. Expressions of angiogenesis and nerve regeneration related proteins can be analyzed by western blotting. We will explore the time-space relationship between angiogenesis and nerve regeneration according to the above mentioned results of observation indices. We hope to develop a feasible technique of controlling release of growth factors (GF) in ASCS via present study, and to establish a new theory/method originated from vascular reconstruction in secondary injury of SCI.

脊髓损伤（SCI）后局部血管重建与继发性损伤严重程度关系密切。但是，以往研究忽视血管化在SCI修复过程中的关键作用，微循环破坏后的血管与神经再生的关系尚未明确。本研究以前期研究为基础，制备封装血管内皮生长因子（VEGF）和神经营养因子-3（NT-3）的脊髓脱细胞支架（ASCS），联合携带绿色荧光蛋白（GFP）的大鼠胚胎脊髓来源神经干细胞（ENSC），在SD大鼠脊髓半切损伤模型中研究持续释放VEGF和NT-3的ASCS的移植时机。Micro-CT观察SCI后脊髓血管重建的3D影像，辅以数据分析、血管密度计算评价血管化程度；免疫组化观察干细胞分化及轴突、髓鞘再生；透射电镜观察微血管及髓鞘形态；免疫印迹法分析血管、神经相关蛋白的表达，研究血管再生与神经再生的时-空关系。希望通过本研究开发控释生长因子ASCS的成熟技术，并以脊髓继发损伤中的血管重建这一切入点，建立SCI治疗的新理论和新方法。

背景：促进血管再生重建是治疗脊髓损伤（SCI）的潜在靶点。血管内皮生长因子（VEGF）是一种促血管形成因子，被证实具有神经营养和神经保护作用。基于VEGF和神经营养因子-3（NT-3）在血管生成和神经修复中的协同作用，在脊髓损伤部位共释放VEGF和NT-3具有广阔的治疗前景。..方法：采用乳液和溶剂挥发法制备封装VEGF165或NT-3的聚乳酸-羟基乙酸共聚物（PLGA）纳米微球（NPs）（V-NPs或N-NPs）。在脊髓半切损伤大鼠的损伤部位移植负载VEGF的脱细胞脊髓支架（ASCS）（V-ASCS），通过高分辨率的显微CT扫描并分析血管生成和重构情况。随后，深入研究VEGF165和NT-3共释放对大鼠脊髓半切损伤的治疗作用。接种骨髓间充质干细胞（BMSCs）的ASCS移植（ABC组）作为基础治疗方法，联合局部共释放VEGF165和NT-3（ABVN组）作为观察对象。研究中采用免疫荧光染色、组织学染色和Basso，Beattie和Bresnahan（BBB）运动评定量表评价血管再生及神经修复作用。..结果：VEGF165和NT-3封装效率高达(90.8±3.1)%和(90.2±2.6)%。VEGF165释放曲线示，首个24小时，分别快速释放1.966μgmgNPs-1和1.045μgmgV-ASCS-1。持续释放阶段，每天约0.040μgmgNPs-1和0.022μgmgV-ASCS-1至720h。NT-3释放曲线也分为初始快速释放，相对较快的释放和持续释放阶段。在大鼠脊髓半切模型中，V-ASCS在损伤部位的植入显示了对血管生成和重构的促进。组织学证实了该结果。然而，BBB评分观察到的运动功能改善是有限的。内皮细胞RECA1免疫荧光染色表明共释放有助于促进损伤后1周局部微循环重建，而8周无明显影响。神经元，轴突和星形胶质细胞相关生物标志物（NeuN，NF -H和GFAP）荧光染色表明ABVN组更好地保存了神经元，同时抑制胶质瘢痕形成。HE、神经元/髓鞘染色证实了ABVN组更优的组织学改善。BBB评分证实了共释放对运动功能的改善作用。..结论：缓释VEGF的ASCS移植加快SCI大鼠的血管重建进程。共释放VEGF165和NT-3强化了ASCS/ BMSCs移植对SCI大鼠的基础治疗作用。