控释型纳米级5-FU醇脂体抑制周围神经瘢痕增生的效果评价

In order to develop a peripheral nerve regeneration path and to obtain sufficient functional recovery after nerve repair,it is important to remove the physical obstacles which cause neural scarring to interfere with the extension of newly regenerated neurites as well as to avoid chronic persistent neurologic symptoms because neural scarring is at the site of peripheral nerve repair during the nerve recovery. We have prepared a nano-ethosomal carrying 5-Fluorouracil that can go through the intercellular space of hypertrophic scar fibroblasts. Based on this and with the aid of the 5-Fluorouracil ethosome nanometer scale effect, the changes of biological characteristics and ultramicrostructure of primary fibroblast of neural scarring will be evaluated by Live Cell Station methods after treatment by nano-ethosomal encapsulated with 5-Fluorouracil. In addition,the nano-ethosomal carried 5-Fluorouracil will be applied to the scar area of the rat sciatic nerve by a Micro Infusion Implanted Pump(MIIP) in a time and dose controlled manner in order to show a favorable effect during the repair in nerve regeneration. Nerve to Conduction Velocity(NCV) and Compound Muscle Action Potential(CMAP) will be tested by a Electromyography(EMG) machine. Fibroblast morphological changes at different periods of nerve regeneration as well as the axon density, axon diameter, myelin thickness , G ratio and Scar Elevation Index(SEI) will be studied and calculated by double-labelling immunofluorescence, Laser Scanning Microscope(LSM) and Image Pro Plus software,etc. In addition, myelin proteins expression such as myelin protein zero(P0) and peripheral myelin protein (PMP22) will be determined by Western Blot quantitatively. In all, this program will address the role nano-ethosomal carried 5-Fluorouracil in reducing neural scar formation and adhesion after sciatic nerve repair in rats,and explain the possible mechanism in relieving secondary nerve injury during the regenerated process. It is very likely to offer a new window to improving the microenviornment of peripheral nerve regeneation.

减少周围神经轴索延伸途中神经瘢痕物理性障碍，避免再生纤维由神经瘢痕导致的继发损伤，是重建周围神经再生通路，提高神经功能康复的关键和新途径。我们在前期实验已成功制备可通过皮肤瘢痕成纤维细胞间隙的纳米级载5-氟尿嘧啶（5-FU）醇脂体。本项目在此基础上进一步完善其理化特性，凸显纳米尺度效应，通过活细胞工作站等方法观测其对神经瘢痕原代成纤维细胞生物学特性及超微结构的影响；采用控释型植入式微量注射泵将其作用于大鼠坐骨神经损伤模型，通过神经电生理检测再生神经功能恢复状态；结合免疫荧光，共聚焦，Western等形态学及分子生物学方法，检测神经再生修复不同阶段，吻合口再生髓鞘形态，轴突直径密度，G值，神经瘢痕指数及髓鞘蛋白P0，PMP22表达变化；阐明其在神经再生修复阶段持续抑制吻合口瘢痕增生的效果以及对再生神经继发损伤的影响和可能作用机制，为改善周围神经再生微环境及神经损伤康复提供新切入点。

本项目通过构建及表征纳米级醇脂体载抗瘢痕药物5-氟尿嘧啶（5-Fluorouracil,5-FU）理化特性，验证前期实验即纳米级脂质体药物载体借助纳米尺度空间改变脂质体通常的空间构像，促使其通过与细胞膜相似相融进入瘢痕组织细胞。本项目“控释型纳米级5-FU醇脂体抑制周围神经瘢痕增生的效果评价”，将不同浓度纳米级5-FU醇脂体作用于原代神经瘢痕成纤维细胞，观测其对神经瘢痕成纤维细胞的活性以及分泌的I型-胶原蛋白的表达变化；得出0.03%浓度的纳米级5-FU醇脂体效果最为明显（P<0.05）；本项目还创新性地引入控释型植入式微量注射泵，应用微量注射泵定时定量将纳米级醇脂体载5-FU作用于大鼠坐骨神经损伤瘢痕组织，运用分子生物学方法系统、量化的评价其对再生神经纤维瘢痕组织的形态学及功能影响，借助透射电镜及免疫组化法观测神经吻合口细胞超微结构及再生神经纤维形态变化、通过Western blot等方法，检测神经髓鞘蛋白等细胞因子分泌变化，进一步验证0.03%(w/v）的纳米级醇脂体载能够抑制神经吻合口瘢痕细胞分泌，与对照组相比，效果明显；应用0.03%浓度的纳米级5-FU醇脂体于大鼠坐骨神经离断模型抑制神经瘢痕增生，通过对大鼠足迹行为学检测，腓肠肌湿重，坐骨神经功能指数，显示与对照组存在显著差异（P<0.05）。通过本项目实验结果得出，0.03%浓度的纳米级5-FU醇脂体抑制神经瘢痕组织及细胞增生，减少轴索延伸的物理性障碍促进再生神经纤维延伸的作用。本研究探讨控释型0.03%(w/v）纳米级5-FU醇脂体对神经瘢痕增生的抑制效果，为改善周围神经再生微环境提供新思路以及开发新药物提供理论基础和实验依据。目前已发表标注该课题资助的SCI论文5篇，其中项目负责人作为第一作者，发表在international Journal of Molecular Sciences.2014, 15(12)，22786-22800)；项目负责人作为通讯作者，发表在Biomaterials. 2016, 21;103: 256-264。