含TNF-α抑制剂缓释系统神经移植物的制备及其修复大鼠坐骨神经10mm缺损的研究

Tissue engineered nerve grafts (TENGs) represent a promising alternative to autologous nerve grafts used for peripheral nerve repair, and have become a focus in research of peripheral nerve regeneration. The results of my project that has been previously sponsored by National Natural Science Foundation (grant No. 81000678) showed that the time window of nerve regeneration is a key point for the regeneration outcome. Therefore, we concern about on molecular events occurring at an early stage following peripheral nerve injury and their correlation with regeneration mechanisms, and attempt to develop new breakthrough in research of peripheral nerve regeneration. In preliminary experiments, whole rat genome microarray has indicated that a number of survival/ apoptosis genes are related to tumor necrosis factor receptor1 (TNFR1), and ELISA assay found that tumor necrosis factor-α (TNF-α) protein level increased after nerve injury. These findings collectively suggested the dose-dependent dual effects of TNF-α on primary cultured Schwann cells, i.e., an increase and decreased cell proliferation and migration or reduced and enhanced cell apoptosis induced by lower and higher dosages of TNF-α, respectively. Based on this premise, this project aims to construct a novel TENG that is featured by a controlled release drug delivery system through immobilizing a TNF-α inhibitor to a silk fibroin-based nerve conduit by using genipin as a crosslinking agent, and to use the TENG for repairing a 10 mm long sciatic nerve defect in rats. Our design is hoped to optimize an immune microenvironment by controlling the dosages of TNF-α used, and thus to inhibit apoptosis of Schwann cells and accelerate nerve growth, thus leading to the better outcome of nerve regeneration and function restoration.

寻找自体神经替代物修复周围神经缺损，一直是周围神经再生研究热点。申请者前一个国家自然科学基金项目发现，再生的时间窗口是决定再生结果的关键。鉴于此申请者开始关注从损伤早期以及周围神经再生机制上，寻求周围神经损伤修复研究的新突破。大鼠全基因组芯片检测结果发现，多个调控生长与凋亡相关的差异表达基因均与TNF-α受体1有关。ELISA检测坐骨神经离断后，TNF-α蛋白表达量逐渐增加。并且TNF-α在不同剂量下对体外培养施万细胞，呈现双向调控作用，即低浓度有促施万细胞增殖作用；而高浓度则抑制施万细胞增殖迁移，并最终引发凋亡。本研究申请者拟运用组织工程研究手段，构建含TNF-α抑制剂缓释系统的神经移植物，并将其用于修复大鼠坐骨神经10mm缺损。通过调控周围神经损伤后局部TNF-α浓度，以期改善局部免疫微环境，抑制施万细胞凋亡，加快神经生长速度，促进神经功能良好恢复，为周围神经损伤修复提供新思路新方法。

周围神经损伤导致肢体麻痹、瘫痪以及丧失对相应身体区域自主控制等一系列症状。临床上以移植自体神经为主要治疗手段，由于其具有诸多弊端，近年来构建组织工程化神经以修复周围神经缺损，成为周围神经再生领域的研究热点。TNF-α是机体受到伤害性刺激后，巨噬细胞和施万细胞产生的启动整个免疫级联反应的上游炎症因子之一。本课题组前期研究工作发现，不同剂量TNF-α对体外培养施万细胞呈双向调控作用，即低浓度2ng/ml的TNF-α有促施万细胞增殖作用；而高浓度40ng/ml时，则能抑制施万细胞增殖和迁移，且最终引发凋亡。本研究通过生物交联剂京尼平，将TNF-α抑制剂交联固定于壳聚糖导管支架上，构建含TNF-α抑制剂的组织工程化神经移植物。高效液相检测神经移植物中TNF-α抑制剂的体外释放规律，发现其随时间呈缓慢释放；免疫细胞化学染色发现神经移植物浸出液对施万细胞凋亡有抑制作用；扫描电镜观察神经移植物内表面和横截面超微结构，发现其特征有利于再生神经生长以及损伤局部管壁内外物质交换。进一步将该构建成功的组织工程化神经移植物用于修复大鼠坐骨神经缺损。ELISA检测神经移植物中TNF-α抑制剂体内释放情况，显示其在体14天仍能有效释放，并且随时间呈梯度缓慢释放。术后12周，电生理检测再生神经复合肌动作电位和神经传导速度等功能学恢复情况；透射电镜观察再生神经髓鞘厚度和新生髓鞘板层数，肌肉三色染色观察靶肌肌纤维面积以及胶原纤维所占百分数等体内形态学结果，提示组织工程化神经移植物组与阳性对照自体神经组相比没有显著差异，均明显优于阴性对照单纯壳聚糖导管组。值得注意的是，再生早期关键时期4周，组织工程化神经移植物组再生神经纤维所占面积百分比，已显著优于壳聚糖导管组，与自体神经组没有明显差异。本研究成功构建了含TNF-α抑制剂组织工程化神经移植物，通过改善损伤局部免疫微环境，加快了神经生长速度，促进了神经功能良好恢复，为临床治疗周围神经损伤提供了新思路新方法。