应用自体肌外膜仿生神经导管修复周围神经缺损及其机制的实验研究

Preparation of ideal nerve conduits with proper biomaterials is a high priority in peripheral nerve regeneration research. During the past several decades, various types of materials have been used to prepare nerve conduits with different degrees of success. However, all of these materials have shortcomings. The epimysium, the outer layer of dense connective tissue of skeletal muscle, and the epineurium have similar structure, components and function. Thus, the present study employed microsurgical techniques to fabricate tube-like structures, termed autologous epimysium conduit (EMC), by rolling up epimysium containing a few muscle fibers for the repair of peripheral nerve defects. In the preliminary experiment, we succeeded in repairing 5mm mice sciatic nerve gaps with EMCs. And interestingly, we also noticed that some cell components of AECs, which were harvested from the external oblique muscle of EGFP-positive mice, migrated into nerve regeneration area and participated in sciatic nerve regeneration. To assess the feasibility and mechanism of EMC further,an experiment is designed, in which EMCs harvested from C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb mice are prepared to repair sciatic nerve defects of the C57BL/6 mice. At various time points, the structure and function of regenerative nerves will be detected systemically to evaluate AEC effectiveness. During the process of nerve regeneration, the composition and function of cell components and micro blood vessels infrom EMCs will also be analysed. The current study can provide not only a new way about how to use skeletal muscle reasonably and effectively. Meanwhile, it will also perfect the relative mechanisms on how EMCs promote nerve regeneration.

选择合适的材料制备理想的神经导管是周围神经再生研究领域中的重要内容,然而目前所能选 择的材料均各有利弊。肌外膜是肌肉的保护性组织,有着与周围神经外膜相似的结构、成分和功能。因此本课题组首次尝试应用肌外膜组织薄片卷曲制备成中空的自体肌外膜神经导管(EMC),并在预实验中成功修复了小鼠坐骨神经缺损;同时还意外地发现,EMC中的细胞成分进入神经再生区域并参与了再生神经的结构重建。为了进一步明确EMC的应用可行性及EMC中细胞成分参与神经再生的作用机制,我们拟将雄性CAG-EGFP转基因小鼠的EMC桥接修复同源野生型雌性小鼠坐骨神经缺损,并在12周后通过神经电生理、组织学检测、肌肉功能检测等手段系统地 评测再生神经结构和功能,同时对参与神经再生的细胞种类及其参与形式进行分析。本研究将为合理、高效地利用肌源性组织促周围再生提供一种新方法，同时扩展并完善了活体肌源性组织促再生的相关机制。

探讨腹外斜肌外膜制备的肌外膜导管（epimysium conduit，EMC）中的细胞成分在修复小鼠坐骨神经长段缺损中的作用。本研究分别设立：普通肌外膜导管组，无特殊额外处理；辐照组，将肌外膜进行辐照抑制管内活细胞迁移；脱细胞组，通过细胞灭活方式消除管内活细胞的生物活性。选取8周龄雄性 C57BL/6J 增强绿色荧光蛋白（enhanced green fluorescent protein，EGFP）小鼠的腹外斜肌，修剪并制备成EMC。采用不同剂量X线（0、15、25、 35、45、55、65 Gy）进行细胞迁移抑制处理，选择合适剂量；采用脱氧胆酸钠方法行脱细胞处理，常规 HE 和马松染色鉴定脱细胞效果后制备 EMC。共采取24 只 C57BL/6J 野生型小鼠制备右后肢 5 mm 长坐骨神经缺损模型后，分别采用 EMC、辐照抑制 EMC、细胞灭活EMC修复缺损。术后8、12周取再生神经中段行大体观察、甲苯胺蓝染色、免疫荧光染色以及透射电镜观察，同时，切取同侧腓肠肌，对比腓肠肌重量，评估神经功能恢复情况。肌外膜细胞迁移随照射剂量增大，细胞迁移数逐渐减少，35 Gy后光镜下无明显细胞迁移；各剂量组较辐照前细胞迁移数量差异均有统计学意义（P<0.05），最终选用经 35 Gy 照射剂量处理后续的体内实验。脱氧胆酸钠脱细胞法对肌外膜纤维损伤最小。在体内实验中，术后12周各组 EMC中神经均再通，正常肌外膜组中修复后的坐骨神经最粗，细胞抑制组次之，脱细胞组最细；免疫荧光染色可见，正常肌外膜组 EMC中 EGFP 细胞包绕再生轴突；甲苯胺蓝及透射电镜观察，正常肌外膜组再生神经轴突数及再生有髓神经鞘厚度显著优于其余两组（P<0.05），且此两组间比较差异无统计学意义（P>0.05）。在神经功能评估上，正常肌外膜组的腓肠肌湿重明显大于其余两组。且血管检测提示，EMC中原微血管结构可能在移植早期及实现了血运的再通，有利于周围神经的再生修复。这些结果揭示了小鼠腹外斜肌中的细胞成分参与并促进了小鼠坐骨神经的再生。本研究将为合理、高效地利用肌源性组织促周围再生提供一种新方法，同时扩展并完善了活体肌源性组织促再生的相关机制。