【作者】 尤凤健；

【导师】 罗卓荆；

【作者基本信息】 第四军医大学 ， 外科学， 2011， 硕士

【摘要】 周围神经损伤是临床上常见和严重的创伤并发症之一。目前,受损神经的再生及功能修复仍是一项尚未解决的临床难题。对于短节段的神经缺损,可直接将其端-端吻合,使近端再生的神经纤维能够穿过受损区长入远端。但长节段的神经缺损,往往需要通过神经移植术进行治疗。自体神经移植仍然是目前治疗长节段神经缺损的“金标准”。然而,自体神经移植受到很多因素限制,如:供区神经来源不足,需要手术获取供体神经,继发供体神经支配区肢体部分功能丧失以及供受神经在组织结构等方面不匹配等。因此,寻找能够替代自体神经移植的方法是目前周围神经修复领域亟待解决的难题。随着组织工程研究逐渐成为研究的热点,应用组织工程学方法构建组织工程神经支架来桥接长节段神经缺损收到比较满意的效果。随着研究的不断深入,越来越多的具有高仿真结构的神经支架被报道,然而,仅有少数关于复合种子细胞神经支架试验研究的文献报道。本课题选用胶原为主要原料,应用冷冻干燥技术,构建出在组成成分及内部结构等方面均与正常神经高度相似的具有轴向微管样结构的三维多孔支架材料。笔者又选用具有来源广、易培养且具有多向分化能力等诸多优点的骨髓基质干细胞与其复合。利用多种形态学及功能学的研究方法综合评估该复合支架桥接大鼠15mm坐骨神经缺损的修复效果,并与自体神经移植及单纯支架移植比较,结果证实其疗效接近自体神经移植并优于单纯支架移植。具体内容如下:第一部分骨髓基质干细胞的培养及鉴定目的:探究大鼠骨髓基质干细胞的培养方法及生长条件,为其与胶原-壳聚糖神经支架复合并修复大鼠周围神经缺损奠定基础。方法:取体重约70g左右的SD大鼠,体外培养骨髓基质干细胞。倒置相差显微镜下观察原代、传代及诱导分化细胞的生长情况及形态学改变,并描绘生长曲线。结果:细胞贴壁生长并以细胞集落的方式增殖。培养至第3代的细胞呈分布均匀的纺锤形细胞生长。可通过简单方法诱导其向神经细胞及成骨细胞方向分化。结论:培养至第3代的骨髓基质干细胞具有较高的细胞纯度,且具有良好的干细胞分化活性,可作为组织工程种子细胞应用。第二部分胶原-壳聚糖神经支架的构建及其与骨髓基质干细胞的复合目的:构建一种组织工程神经支架,并观察体外培养的骨髓基质干细胞在其内部的生长情况,为种子细胞的移植提供阶段性实验数据。方法:以Ⅰ型胶原蛋白和壳聚糖为原料通过冷冻干燥技术构建神经支架,扫描电镜观察其内部结构,测量其孔径大小、孔隙率等指标。将体外培养的骨髓基质干细胞与Ⅰ型胶原蛋白-壳聚糖神经支架复合,共培养3天;扫描电镜观察细胞在支架内部的生长情况。结果:构建出的神经支架均为圆柱状,内部呈纵向平行排列的微管样结构,且孔径均匀。细胞贴附在支架微管内壁上,且生长状况良好。结论:Ⅰ型胶原蛋白-壳聚糖支架具有良好的内部三维结构和生物相容性,可与细胞复合后用于修复周围神经缺损。第三部分复合神经支架修复大鼠坐骨神经缺损的有效性评估目的:利用体内试验综合评估复合神经支架修复周围神经缺损的有效性。方法:应用免疫组织学、透射电镜观察、神经电生理检测、逆行示踪标记以及计算坐骨神经指数等研究方法,从形态学和功能学两方面综合评估复合神经支架修复大鼠15mm坐骨神经缺损的效果。结果:神经支架在体内能够保持其完整结构大约4周。复合支架材料组、单纯支架材料组修复效果均接近自体神经移植,复合支架材料组稍优,有大量再生神经纤维长入远端。荧光显微镜观察结果显示,支架几乎完全降解,并被大量再生神经纤维替代(经京尼平交联后,本支架自发红色荧光)。修复段可见轴向微管间有NF160及S-100阳性的再生神经纤维穿过,证实再生的神经纤维通过了修复段。透射电镜结果:新生的髓鞘比较纤细,结构完整,有大量无髓神经纤维和有髓神经纤维共存,可观察到板层结构良好的髓鞘,再生神经轴突情况良好,修复效果接近自体神经移植。功能学检测结果显示:复合支架组的运动神经传导速度、潜伏期和波幅与自体神经移植结果接近,两组之间差异无统计学意义(p>0.05);荧光金标记后,观察到阳性神经元的数量在脊髓前角和背根神经节等部位均与自体神经移植组相当;坐骨神经指数结果也同样证实,复合神经支架桥接修复坐骨神经缺损,其功能性恢复效果接近自体神经移植,并优于单纯胶原-壳聚糖支架组。更多还原

【Abstract】 Peripheral nerve injury is a common and serious disease exposed to physicalinjuries; the regeneration of injury nerve and its functional recovery are achallenge to current clinical. For short nerve gaps that there is no nerve tissue lossor possible approximation with minimal tension, we choose direct neurorrhaphyto suture the divided nerve through end-to-end or end-to-side coaptations. But forlong nerve gaps, we can’t coaptate the divided nerve directly. Currently, we takeautologous nerve grafting as the gold standard treatment. But there are someinherent drawbacks limit autologous nerve grafting, such as limited availability ofdonor nerves, the need for a second surgery to obtain the donor nerve, donor sitemorbidity and secondary deformities, as well as mismatch between the injurednerve and the donor nerve, etc. So seeking promising alternatives to supplementor even substitute autologous nerve grafts is still a major challenge to peripheralnerve repair.In recent years, using tissue engineering grafts to bridge long gaps has produced promising results and gained extensive attention. With the deepeningresearch of tissue engineering, more and more high-simulation structure neuralscaffolds were reported. However, only few studies about compounds seed-cellswere reported.In this topic we choose collagen as the main material, and by freeze-dryingtechnology we successfully fabricated a collagen-chitosan scaffold which with aninterconnected porous structure and longitudinally oriented pore channels. Andwe choose bone marrow stem cells which were source wide, easy to cultivate andhave the ability to multiplex differentiation to combine with it. Finally, we use thecombined nerve scaffold to bridge a 15 mm long sciatic nerve defect in rats andevaluate its efficacy by using a combination of morphological and functionaltechniques. Specific content as follows:Part one: Culture and Differentiation of Bone marrow stem cells.[Objectives] To study the growth condition and training methods of rats bonemarrow stem cells for laying the foundation of combining with bone marrowstem cells and repairing rat peripheral nerve defects.[Methods] Taking weight around 70g SD rats to culture bone marrow stem cellsin vitro. Growth conditions and morphological characteristics of primary cells,passage cells and differentiated cells were monitored by an inverted phasecontrast microscope. And we also depict the growth curve.[Results] Cells grow in an adherent way and proliferate by the cell colony way.The 3rd generation cells are growing like a spindle, and can be induced intoneural cells and osteoblasts through simple methods.[Conclusion] The third generation of bone marrow stem cells have higher cellpurity and good stem cell activity, can be used as seed cells of tissue engineering. [Objectives] Developing a tissue engineering nerve scaffold, and observe thegrowth of bone marrow stem cells cultured in vitro on the scaffold, to provide thestage-experiment data for the transplantation of seed cells.[Methods] The scaffold was made of I-collagen and chitosan by freeze-dryingtechnology. Using scanning electronic microscope to observe there coursedirections and measure the size of the micropores and the factor of porosity. Thebone marrow stem cells were seeded on the collagen scaffolds and cultured for 3days, and then observe the growth of bone marrow stem cells cultured in thescaffold.[Results] All scaffolds were circular cylinder, the microscopic channels werearranged in parallel manners, and the pore sizes of the channels were uniform.The bone marrow stem cells were seeded in the scaffolds successfully.[Conclusion] I-collagen-chitosan scaffolds have good structure andbiocompatibility, and can be used for repairing the nerve injuries after combinedwith bone marrow stem cells.Part three: The effectiveness of combined collagen-chitosan scaffolds inbridging 15 mm long sciatic nerve defect in rats[Objectives] To investigate the effectiveness of combined collagen-chitosanscaffolds in bridging 15 mm long nerve gap in rats.[Methods] Using combination of morphological and functional techniques,including TEM, retrograde-labelling, immunohistology, electrophysiology andbehavioral tests, to investigate the effectiveness.[Results] After implantation, the nerve scaffold can maintained its microstructureintegrity for about four weeks. The results show that compound scaffold group and the pure scaffold group are close to autologous nerve grafts, compoundscaffold group little good, a large number of regenerative nerve fibers grow intothe remote. Fluorescence microscopy showed that scaffolds were almostcompletely degraded and were replaced by many regenerated nerve fibers(aftercross-linked by genipin,the scaffold spontaneous red fluorescence). TEM Results:there are many unmyelinated and myelinated nerve fibers co-exist. The newmyelin is thin but has integrity structure and well-structured lamellar. Functionresults: the compound scaffold group’s motor nerve conduction velocity, latencyand amplitude are close to nerve autograft and no significant difference betweenthe two groups (p>0.05); FG labeled tracer shows that the number of positiveneurons which can be detected in the spinal anterior horn and dorsal root gangliais not far off auto-graft. The SFI results also confirm that the functional recoveryeffect of the compound nerve scaffold is close to nerve autograft, and better thanpure scaffold group.Bone marrow stem cells; Collagen; Scaffold更多还原