【作者】 张彩顺；

【导师】 吕刚；

【作者基本信息】 中国医科大学 ， 外科学， 2007， 博士

【摘要】 组织工程人工神经移植物修复大鼠坐骨神经缺损的实验研究前言外伤造成周围神经缺损在临床中很常见，要想恢复神经功能必须恢复神经的连续性。对于短距离的缺损(＜3cm)可以通过游离神经、神经改道或屈曲关节等方法利用显微外科技术将神经两断端直接连接起来；而对于较长距离的缺损(＞3cm)则需要一桥接物将神经断端连接起来。到目前为止，自体神经被认为是修复长距离神经缺损的最佳方法；但是由于自体神经来源受限，且会导致供区感觉神经新的功能丧失，而且感觉神经细小，无法满足较粗神经缺损的需要等缺点限制了其在临床上的广泛应用。因此，人们一直在努力寻找一种新的自体神经替代物来桥接神经缺损。同种异体神经与自体神经最为相似，且来源丰富，但由于存在免疫排斥反应，需要长期应用免疫抑制剂而限制了其临床应用；如何降低移植神经的抗原性是目前研究的热点。研究表明-196℃液氮低温冷冻后的神经移植，抗原性降低，免疫反应较轻，但仍难以达到临床应用的要求。在免疫排斥反应得到很好解决之前，异体神经移植尚难广泛应用。其他自体非神经材料：动脉、静脉、筋膜、骨骼肌等桥接神经缺损虽然在动物实验取得一定成功，但应用到临床还有很大差距。随着材料科学和工程技术的发展，硅胶管、PGA、PLA、聚丙烯、胶原、PLA/PGA、聚丙烯/聚丙烯氰共聚物等多种高分子人工合成材料也应用于修复周围神经缺损，但效果都不理想。二十世纪80年代兴起的组织工程学为周围神经缺损的修复提供了新的研究方向。组织工程学(Tissue Engineering)是一门以细胞生物学和材料学相结合，进行体外或体内构造组织或器官的新兴学科。其基本原理为：从机体获取少量的活性组织，用特殊的酶或其他方法将细胞(又称种子细胞)从组织中分离出来并在体外进行培养扩增，然后将扩增的细胞与具有良好生物相容性、可降解和可吸收的生物材料按一定的比例混合，使细胞粘附在生物材料上形成细胞一材料复合物；再将该复合物植入机体的组织或器官病损部位，随着生物材料在体内逐渐被降解和吸收，植入的细胞在体内不断增殖并分泌细胞外基质，最终形成相应的组织或器官，从而达到修复创伤和重建功能的目的。周围神经组织工程学研究的内容就是将具有促进周围神经再生的种子细胞种植于天然或人工合成材料制备的支架中，用来桥接周围神经长距离缺损。组织工程的三大要素包括种子细胞、生物材料及体内微环境。与传统的生物替代材料修复组织方法不同，组织工程技术特别注重将种子细胞与生物材料复合，形成与自身组织有着同样结构和功能的生物组织以修复组织缺损。因此，种子细胞在这三大要素中起着关键的作用。随着人们对神经再生机制研究的不断深入，人们发现施万细胞(Schwann cells，SCs)在周围神经再生中起重要作用；神经损伤后SCs分裂，增殖形成Bungner带，并分泌NGF等神经营养因子，引导近端轴突向远端生长；因此，SCs被认为是周围神经组织工程的种子细胞。但由于自体SCs需预先切取自体神经分离培养，给病人带来新的创伤，且体外扩增速度慢、细胞纯度低，难以达到组织工程学要求的数量。骨髓间充质干细胞(Bone Mesenchymal Stem cell，BMSc)是存在于骨髓中的一种具有多向分化潜能的干细胞。经实验证实体外培养的BMSc可以在体内、体外向神经细胞、神经胶质细胞分化，并能促进周围神经损伤轴突的生长；并且自体BMSc易于获取并能在体外迅速扩增获得足够数量的细胞用于组织工程构建且不易造成供区部位的创伤，克服了自体SCs来源受限，体外扩增速度慢，细胞数量少等缺点；目前BMSc作为组织工程新的种子细胞已被应用于其他组织的构建和组织缺损的修复。良好的生物材料是周围神经组织工程的支架。随着高分子材料科学的发展，出现了多种适合神经再生的生物材料，如PGA、PLA等；但这些材料只在一定程度上能促进神经再生，尚不能满足神经组织工程的要求；良好的神经组织工程支架材料必须具备：①良好的生物相容性；②足够的力学性能和良好的生物机械性能；③良好的生物稳定性；④溶出物及可渗出物含量低；另外还需在结构上类似神经使种子细胞自然粘附，形成Bungner带，引导神经再生。我们应用化学方法处理同种异体神经，成功地制备了无细胞同种异体神经支架；该支架具有天然的三维立体空间结构，可以更好地提供种子细胞粘附、生长。本实验将体外培养、扩增的BMSc种植入具有天然立体空间结构的无细胞神经支架上，桥接大鼠坐骨神经15mm缺损，研究该移植物对神经功能恢复的修复效果，为组织工程周围神经的进一步研究和临床应用提供实验依据。实验方法应用化学方法制备无细胞神经移植物；体外培养扩增BMSc，传至三代，待细胞纯化后，Brdu标记。将标记的BMSc注入无细胞神经移植物后复合培养7d，观察细胞与支架结合情况；在分别利用种入BMSc及培养液的无细胞神经移植物和自体神经桥接接大鼠坐骨神经15mm缺损；术后12W行大体形态观察、胫骨前肌湿重比、神经电生理、腓肠肌透射电镜、足底皮肤S-100检测、HRP逆行标记等方法评价神经修复效果。实验结果1、应用改良方法制备的无细胞神经移植物周期仅为11天；正常神经内的SCs、髓鞘、轴索均消失，仅剩下SCs基底膜管，基本消除了抗原成分；经Laminin免疫组化染色证实基底膜管内存在Laminin，保留了具有引导促进轴突生长，并且具有粘附细胞作用的层粘连蛋白。2、体外培养的BMSc传至三代，细胞呈长梭形，螺旋状排列于瓶底，免疫组化染色证实为非造血干细胞；扫描电镜观察BMSc均匀分布于无细胞神经移植物管腔内，细胞呈椭圆形或多突起形，伸出伪足与管壁紧密相连。3、实验组大鼠术后12W手术侧足址可以分开，并能支撑着地；足底皮肤S-100蛋白染色阳性；HRP逆行标记到达后根神经节及脊髓；胫骨前肌湿重比、神经传导速度、髓鞘厚度、神经纤维直径与自体神经移植组相比差异无显著性，与空白对照组具有显著性差异。结论1、无细胞神经移植物消除了细胞等抗原成分，保留了SCs基底膜管，具有天然立体三维空间结构，可以作为组织工程神经支架。2、无细胞神经移植物在制备过程中保留了Laminin，可以引导促进轴突生长。3、BMSc与无细胞神经移植物具有良好的细胞亲和性，两者可以复合构建组织工程人工神经桥接周围神经缺损。4、BMSc与无细胞神经移植物复合构建的人工神经移植物可以促进周围神经再生和功能恢复。5、足底皮肤s-100蛋白染色可以评价感觉功能的恢复。更多还原

【Abstract】 PREFACEPeripheral nerve defects caused by trauma are encountered commonly in clinic. If you want to recover function of the nerve you must restore their continuity. We can utilize microsurgical technique to connecte the nerve stump directly by separate nerve, change the path of nerve or flex articulation ect for nerve defects of <3cm.But the long gaps ( > 3cm) must be connected by graft. At present,autologous nerve transplantation has been considered the best method to repairing the long nerve defect. Because of the limited availability and donor-site morbidity of autograft,the mismatch of nerve cable dimensions between the donor graft and the recipent nerve ect, autologous nerve transplantation cannot be applied in clinic extensively. So many researchers concentrated on searching for a new graft to replace these autografts. Allograft represent a reconstructive alterative ,but are limited because of great rejection and the need for long-term immunosuppression. A study has shown that allografts freezed by—196℃decrease their antigon and immune response,but it cannot meet clinical needs. Other non-nerve tissues such as arteries,veins,muscle ect ,were used as alternatives to suture repair of nerve to successfully bridge nerve gaps in animal experiment. But there is a large gap to use in clinic. With the development of material science and engineering technique,artificial synthetic materials such as silicone tube ,PGA,PLA ect, have been used for repairing peripheral nerve defects,but the functional recovery is not satisfactory.With the emergement of tissue engineering in 1980s,there is a new project in using tissue engineering approaches to bridge peripheral nerve defects. Tissue engineering is formally defined as the application of the principles and methods of engineering and the life science toward the fundamental mammalian tissues and the development of biological subsitutes that restore,maintain,or improve tissue function. The principle of tissue engineering is :a small number of cells can be harvested from the patient using a biopsy ,and then the harvested cells are cultured in vitro in order to get the appropriate number in the laboratory. These cells can then be grown within a biocompatible ,absorbable natural or synthetic scaffold and implant into the defective site. The scaffold degrade gradually,the cells secrete various matrix materials to create an actual living tissue ultimately. The basic principles of tissue engineering of the peripheral nerve is: the cells,which can promote peripheral nerve regeneration, are seeded into natual or synthetic scaffold to bridge peripheral nerve long gaps. A tissue engineering scaffold should provide a necessary mechanical support as well as a physical structure for the transplanted cells to attach,grow, and maintain differentiaed functions. It has shown that Schwann cells display a very imporment role in peripheral nerve regeneration by proliferating and forming bands of Bungner,and releasing neurotrophic factors and directing the proximal axon to distal stump. Although Schwann cells are very effective in inducing nerve regeneration,their clinical use is limited because it is diffcult to obtain a sufficient number of cells to satisfy the requirement of tissue engineering. In the cultivation of Schwann cells used in autotransplantation, another peripheral nerve have to be sacrificed and that frequently leads to multiple surgical procedures and the loss of function at the donor site.Bone Mesenchymal Stem cell(BMSc),which are also know as stromal stem cells,are believed to be multipotent cells that can replicate as undifferentiated cells. Recent experimental studies suggested that cultured BMSc can differentiate into neural cells and glial cells in vitro and vivo, and promote axonal regeneration in peripheral nerve defect. In addition, autologous BMSc are easy to isolate and culture to obtain a sufficient number of cells.Therefore,BMSc overcomed the difficulties encountered with the use of autologous SCs for transplantation,it can be considered to be an alternative to SCs.There are some general requirements for a favourable peripheral nerve tissue enginerring graft: first,the graft should be biocompatible.Second,the graft material should be flexible and strong. Third, it should be permeable. In addition,the intrinsic structure of graft should not only provide a great surface for the attachment of implanted cells,but also imitate the function of bungner bands in which Schwann cell columns were formed to guide nerve regeneration. We have developed a chemical method to treat allograft, acellular nerve allograft was made successfully. This nerve scaffold has an structure of natural three-diamensional space. It may provid a more suitable environment for the transplanted cells to attach ,grow,and maintain differtiated functions. In our experiment,cultured BMSc were microinjected into the scaffold to repair a 15mm defect in the rat sciatic nerve. To investigate the effectiveness of this graft promote functional recovery,and provid experimental support for clinic work and the further study of peripheral nerve tissue engineering.MATERIALS AND METHODSAcellular nerve allograft were made by chemical extraction. The BMSc were isolated and cultured ;It was subcultured for three times and marked by Brdu. BMSc,marked by Brdu,were microinjected into the accelular nerve allograft and cultured for 7 days. The connection of graft and cells were observed by scanning electron microscope. A 15mm defect in the rat sciatic nerve was bridged by accelular nerve allografts filled with BMSc , accelular allograft filled with culture medium solution and autologous nerve respectively. 12 weeks after operation,the effectiveness of functional recovery were assessed by tibial former muscle wet weight comparison ,electrophysiological evaluation, transmission electron microscope of gastrocnemius,immunohistochemical staining of the s-100 protein of foot skin,HRP ect.RESULTS1. The period of making accelular nerve allograft by identified methods is 11 days;The Schwann cells,myelin sheath,axon were disappeared in normal nerve,the basal membrance consuit of SCs only were remained.Laminin,which could promote axon regeneraion and adhere to cells, was observed in basal memberance tubes by immunohistochemical staining.2. Bone Mesenchymal Stem cell performed the long spindle-shaped morphology after subcultured for three times. The scanning electron microscope observation manifested that BMSc show an ellipsoidal shape and attach to tube tightly.3. The tipitoes of experimental rat can separate and support ground 12 weeks after operation.The immunohistochemical staining of the s-100 protein of foot skin is possitive.The HRP was tested in spine and ganglion. Analysis of tibial former muscle wet weight comparison , electrophysiological evaluation,myelin sheath tickness,the diameter of nerve fiber showed no statistically significant difference between experimental group and autograft.CONCLUSION1. The accelular nerve graft eliminated the cells,and remained Schwann cells basal memberance tubes. The scaffold,which three diamensional spsce structure , can be used as tissue engineering graft.2. The Laminin was remained during making accelular nerve graft,which can guide the axon regeneration.3. The affinith of accelular nerve graft to bone marrow stromal cells is good.4. The artificial nerve graft construced by BMSc and accelular nerve graft are able to promote peripheral nerve regeneration and functional recovery.5. The sensory recovery can be tested by immunohistochemical staining of the s-100 protein of foot skin.更多还原