复合红景天苷神经导管的构建及其与MSCs/宿主相互作用的机制研究

Peripheral nerve injuries(PNI) are commonly traumatic lesions , which usually lead to the loss of the sensory and motor function, causing decrease in life quality and affecting patient's physical and mental healths, it is one of the difficult problems in medical science. Applying the nerve relative growth factor as the active ones to fabricate tissue inducible nerve conduit is a focus way to against the disease at present, whereas this methods has some defects, such as instable capabilities of the nerve relative growth factor and easily inactivated, and it is not able to be contented with the demand of clinical treatment. With the financial support from national natural science fund, our previous work on this issue had suggested that salidroside monomer could not only induce mesenechyma stem cells(MSCs) differentiation into nerve and Schwann cells, but also promote differentiated cells secretion nerve relative growth factor, such as nerve growth factor(NGF) and brain derived neurotrophic factor(BDNF). Based on these results, the aim of this study is to adopt the technology of tissue engineering to electrospun collagen/Polycaprolactone (PCL)(1:1) nanofiber scaffold as shell, collagen and chitosan microsphere that encapsulated salidroside as core sheaf to fabricate the combined type nerve conduit for nerve regeneration. The fine mechanisms of the interactions of the behavior of the scaffold with MSCs, will be investigate in the cellular and molecular levels. The characterization of the formation in the specific functional surface, and the mechanism of MSCs and material that participate in neurotization and reconditioning of the host in the special tissue microenvironment will be revealed as well. This item is a novel trial and innovation for the nerve repairing material, which can develop the technology with proprietary intellectual property rights and hopefully can be used in clinic in the future.

各种创伤造成的外周神经损伤修复及其功能恢复,一直是临床医学面临的难题之一。利用神经相关生长因子构建组织诱导性神经导管，是目前研究的热点，但存在外源活性神经生长因子性能不稳定、易失活等不足，尚不能完全满足临床治疗需要。 在国家自然科学基金资助下，课题组前期研究发现，红景天苷单体不仅能促进间充质干细胞（MSCs）分泌神经相关生长因子，而且能诱导其向神经元样和施旺样细胞定向分化。在此基础上，本项目拟利用静电纺丝技术构建以胶原/聚己内酯为壳层，复合红景天苷缓释微球的胶原为芯层的多孔道复合型神经导管材料，从细胞和分子水平探讨导管支架材料的组织诱导性功能及其与MSCs相互作用的响应机制；通过动物实验揭示在宿主特定组织微环境中，材料/宿主间特异性功能界面形成的特征、MSCs及支架材料参与宿主神经再生和修复的机制。该项目的实施，是一种新的尝试和创新，有望形成具有自主知识产权的神经损伤修复材料并用于临床。

外周神经损伤(Peripheral Nerve Injury, PNI)是临床最常见的创伤之一。PNI常常造成神经支配区域退行性病变和功能障碍，已成为世界医学面临的挑战之一。研究具有组织诱导性神经修复材料有望能彻底解决外周神经损伤修复这一临床难。大量研究表明，中药有效成分对外周神经损伤具有修复作用，课题组前期证明红景天苷能诱导MSCs向神经元样细胞定向分化。利用红景天苷构建组织诱导性神经导管并研究其修复功能，是一种新的创新和尝试。.利用三聚磷酸盐方法制备不同含量（10ug/ml、20ug/ml和40ug/ml）红景天苷/壳聚糖微球，微球与聚乳酸-聚羟基乙酸共聚物(PLGA)有机结合制备复合型红景天苷缓释微球。采用特制模具结合冷冻方法制备含不同量红景天苷微球的胶原支架材料芯层；应用静电纺丝技术电纺胶原/PCL复合支架材料的壳层，芯层与壳层嵌套构建复合型神经导管支架材料。分别利用扫描电镜和体外缓释实验，评价微球的结构和缓释效果；采用扫描电镜、体外降解试验、红外光谱技术和力学实验对材料表征；利用CCK-8 方法、HE染色和细胞免疫荧光染色方法，检测大鼠MSCs在支架材料中的粘附、增殖和分化。Wister大鼠30只随机分成5组， 即（胶原/PCL对照组）、（10ug/ml红景天苷材料组）、（20ug/ml红景天苷材料组）、（40ug/ml红景天苷材料组）和自体神经移植组。复制大鼠坐骨神经15mm缺损模型，分别于术后1、3和6月行坐骨神经指数检测，电生理、组织学观察和免疫组织化学检测。结果显示：红景天苷微球具有良好的药物缓释效果；构建的复合支架材料具有多孔性、吸水性、一定力学强度和柔韧性；细胞与材料复合能促进细胞的粘附、生长和增殖，同时能促进细胞向血旺氏样细胞分化；复合红景天苷材料的神经导管坐骨神经功能指数显著高对照组（P<0.05）；潜伏期/传导速度显著低于对照组（P<0.05）；组织学结果显示，复合红景天苷材料的神经导管组再生神经纤维明显高于对照组（P<0.05）；再生神经纤维中S-100, 髓鞘脂蛋白0（P0）, 髓鞘碱基蛋白（MBP）和神经纤维(NF) 的表达水平与对照组比较明显增强（P<0.05），其中以20ug/ml红景天苷材料组尤为明显。构建的复合红景天苷支架材料具有良好的药物缓释效果和生物相容性，对外周神经损伤具有较好的修复功能。