神经胶质细胞移植与脊髓再生

脊髓损伤(spinal cord injury,SCI)后,由于在中枢神经系统(central nervous system,CNS)没有能提供断裂轴突有效再生的微环境而使SCI不能痊愈,给伤者留下严重的躯体功能障碍.大多数神经胶质细胞在CNS移植后均能诱导CNS轴突的再生.在早期,人们从外周神经移植、轴突完全再生的成功经验中发现:外周神经系统(peripleral nervous system,PNS)之所以能完全再生,主要是由于神经轴突的髓鞘细胞-雪旺细胞(Schwann cell,SC,神经膜细胞)能有效地增殖形成引导通道,并分泌大量神经营养因子和细胞分子,促使轴突有效再生,功能恢复.而在CNS与SC相一致少突胶质细胞却无此类功能,其增生迁移晚于再生轴突,不能形成轴突迁移的引导通道,导致再生轴突"迷路",并分泌抑制性因子抑制轴突生长;CNS中另一主要胶原细胞-星形胶质细胞在损伤后能较快反应性增生,过度增生形成胶原瘢痕,使已迷路的中枢再生轴突失去有效再生.因此,在SCI区域,能植入一种有能力改变CNS损伤后微环境、促进和诱导SCI轴突有效修复再生的基质,是人们一直努力的目标.应用SC(包括周围神经)、胚胎脑和脊髓组织、少突胶质细胞以及胶质细胞类的空管膜细胞(ependymal cell)[1]和伸展细胞(tanycyte)[2]等移植于SCI内,通过大量实验证实是有效的,但终未能达到类似于SC在PNS中有作用.     

应用组织工程化周围神经修复大鼠坐骨神经缺损的实验研究

目的:探讨组织工程化周围神经修复大鼠坐骨神经缺损的可行性。方法:应用预制的组织工程化周围神经修复大鼠坐骨神经15mm缺损。术后12周,分别进行显微解剖观察,再生轴突神经电生理测定,再生轴突生长和成熟情况的组织学观察及其图像分析处理。结果:组织工程化周围神经移植体结构完整,组织相容性良好。组织工程化周围神经移植组与自体神经移植组相比相对动作电位幅值恢复率差异无显著性意义(P>0.05)。再生轴突在组织工程化周围神经移植体内生长良好,并可见较好的髓鞘结构形成。组织工程化周围神经移植组与自体神经移植组相比再生轴突密度恢复率差异无显著性意义(P>0.05)。结论:组织工程化周围神经可修复大鼠坐骨神经15mm缺损。     

应用组织工程化周围神经修复大鼠坐骨神经缺损的实验研究

目的：探讨组织工程化周围神经修复大鼠坐骨神经缺损的可行性。方法：应用预制的组织工程化周围神经修复大鼠坐骨神经15mm缺损。术后12周，分别进行显微解剖观察，再生轴突神经电生理测定，再生轴突生长和成熟情况的组织学观察及其图像分析处理。结果：组织工程化周围神经移植体结构完整，组织相容性良好：组织工程化周围神经移植组与自体神经移植组相比相对动作电位幅值恢复率差异无显著性意义(P&;gt;0．05)。再生轴突在组织工程化周围神经移植体内生长良好，并可见较好的髓鞘结构形成。组织工程化周围神经移植组与自体神经移植组相比再生轴突密度恢复率差异无显著性意义(P&;gt;0．05)。结论：组织工程化周围神经可修复大鼠坐骨神经15mm缺损；     

神经营养因子对大鼠坐股神经再生的影响

为探讨神经营养因子(neurotrophicfacters,NTF)对周围神经损伤修复与再生的影响,用大鼠神经再生室动物模型,应用NTF促进坐股神经再生作用进行了研究。结果实验组(应用NTF)大鼠小腿三头肌湿重明显高于对照组;神经干轴突数目和直径明显高于对照组。说明NTF能明显支持周围神经元的存活,促进其再生,尤其是促进运动神经的修复与再生。     

神经营养因子对大鼠坐骨神经再生的影响

为探讨神经营养因子(neurotrophic facters，NTF)对周围神经损伤修复与再生的影响，用大鼠神经再生室动物模型，应用NTF促进坐股神经再生作用进行了研究。结果实验组(应用NTF)大鼠小肠三头肌湿重明显高于对照组；神经干轴突数目和直径明显高于对照组。说明NTF能明显支持周围神经元的存活，促进其再生，尤其是促进运动神经的修复与再生。     

化学去细胞同种异体神经移植修复大鼠骶1神经缺损

背景:自体神经移植是修复周围神经损伤中最常用的方法。目的:探讨化学去细胞神经修复大鼠骶1神经缺损的效果,以期寻找修复周围神经缺损较为理想的方法和材料。方法:取SD大鼠骶1神经,采用化学去细胞法处理大鼠骶1神经的免疫原性成分,使其成为去细胞神经。将Wistar大鼠右侧骶1神经制成1cm缺损模型,用SD大鼠去细胞同种异体神经移植修复大鼠骶1神经的缺损。结果与结论:与术前相比,术后8周逼尿肌漏尿点降低,膀胱最大容积和膀胱顺应性升高(P<0.05)。神经纤维细丝蛋白染色结果显示,右侧经同种异体神经移植修复的神经吻合口断端被染成绿色,神经纤维排列整齐分布均一,再生神经轴突长入远端神经。左侧正常神经形态均一,神经纤维排列整齐分布均一未见轴突长入远端神经。结果表明,化学去细胞同种异体神经的抗原性明显降低,可修复高等哺乳类动物的周围神经损伤。     

周围神经损伤后不同时间的修复比较

背景:大量实验证明,Bungner带-许旺细胞-基底膜结构是神经再生理想的微环境.这一结构在神经损伤两三周后形成.而在神经损伤数小时后,近断端的神经纤维就开始发芽再生神经纤维开始再生与所需微环境的形成并不同步.目的:观察周围神经损伤后不同时间进行修复的最佳效果.设计、时间及地点:随机对照动物实验,于2007-06/2008-06在哈尔滨医科大学动物实验中心完成.材料:新西兰大白兔20只,随机数字表法分为4组:2周后神经修复组、4周后神经修复组、3个月后神经修复组、即时神经修复组.方法:建立成年新西兰大白兔周围神经损伤模型,即时修复组立即缝合伤口,2周后神经修复组、4周后神经修复组、3个月后神经修复组采用神经两断端分别固定于肌膜上,逐层缝合伤口,2周,4周,3个月后重新打开伤口,在手术显微镜下用10-0无损伤尼龙针线进行外膜缝合修复坐骨神经,缝合伤口.主要观察指标:各组缝合神经段的神经电生理、轴突数、光镜及电镜观察结果.结果:2周后神经修复组神经传导速度慢于4周后神经修复组、3个月后神经修复组(P＜0.01);即时神经修复组与2周后神经修复组差异无显著性意义(P＞0.05).2周后神经修复组效果最好,神经纤维走行正常、排列完好,神经纤维可见血管增生,髓鞘结构较好,许旺细胞功能活跃,新生轴突内微缝密集排列.4周后神经修复组最差,神经纤维数量少、排列紊乱,髓鞘轴突变性明显,大部分神经纤维脱髓鞘崩解,轴突消失,未见再生神经纤维.3个月后神经修复组效果较差,可见较多神经纤维结构破坏,捧列略紊乱,髓鞘和轴突变性较明显,仅见少量再生神经纤维,许旺细胞略少,胞质不发达.即时进行神经修复组效果较好,神经纤维结构破坏不明显,排列整齐,髓鞘和轴突变性轻,神经纤维内见有大量再生髓鞘,许旺细胞明显增多,胞质较发达.2周后神经修复组轴突计数优于其他3组(P＜0.05),4周后神经修复组最少.结论:神经损伤2周后进行神经修复效果优于其他时间点,是周围神经损伤后修复的最佳时机.     

化学去细胞同种异体神经移植修复大鼠骶1神经缺损

背景：自体神经移植是修复周围神经损伤中最常用的方法。目的：探讨化学去细胞神经修复大鼠骶1神经缺损的效果,以期寻找修复周围神经缺损较为理想的方法和材料。方法：取SD大鼠骶1神经,采用化学去细胞法处理大鼠骶1神经的免疫原性成分,使其成为去细胞神经。将Wistar大鼠右侧骶1神经制成1cm缺损模型,用SD大鼠去细胞同种异体神经移植修复大鼠骶1神经的缺损。结果与结论：与术前相比,术后8周逼尿肌漏尿点降低,膀胱最大容积和膀胱顺应性升高（P〈0.05）。神经纤维细丝蛋白染色结果显示,右侧经同种异体神经移植修复的神经吻合口断端被染成绿色,神经纤维排列整齐分布均一,再生神经轴突长入远端神经。左侧正常神经形态均一,神经纤维排列整齐分布均一未见轴突长入远端神经。结果表明,化学去细胞同种异体神经的抗原性明显降低,可修复高等哺乳类动物的周围神经损伤。     

应用人工神经修复大鼠坐骨神经损伤的实验研究

目的观察应用复合碱性成纤维细胞生长因子(bFGF)-壳聚糖导管修复大鼠周围神经损伤的效果。方法成年Wistar大鼠25 只分为假手术组(n=10)、单纯损伤组(n=5)和人工神经修复组(n=10)。假手术组仅暴露坐骨神经5 mm,单纯损伤组暴露坐骨神经并切断,人工神经修复组以复合bFGF-壳聚糖导管桥接缺损。术后对实验动物的坐骨神经进行大体观察,对运动进行行为观察,以及组织化学和免疫组织化学方法评价神经再生情况和靶肌肉恢复情况。结果术后5 周,与单纯损伤组相比,人工神经修复组运动功能有一定程度恢复。人工神经修复组再生的坐骨神经已经通过bFGF-壳聚糖导管越过缺损并与远端相连接。免疫组织化学方法显示,坐骨神经再生段可观察到神经微丝(NF)阳性和S-100 阳性纤维。应用Masson 染色方法,可观察到人工神经修复组腓肠肌去纤维化程度相对于单纯损伤组有明显改善。结论应用bFGF-壳聚糖导管能有效修复周围神经损伤并使大鼠运动功能得到改善。     

高压氧与带血供周围神经移植修复鼠脊髓损伤的研究

目的探索联合应用高压氧与带血供周围神经移植修复脊髓损伤的作用。方法将40只雌性Wistar成年鼠分为A、B两组,A组为单纯带血供的正中神经移植修复脊髓损伤;B组为带血供的正中神经移植后给予高压氧治疗。结果在组织学、神经传导速度及移植神经内再生轴突形态计量分析诸方面,效果明显好于A组。结论带血供周围神经是修复脊髓损伤较为理想的移植材料,高血氧能够提高带血供周围神经移植修复脊髓损伤的能力。     

A new type of Schwann cell graft transplantation to promote optic nerve regeneration in adult rats

Like other parts of the central nervous system, the adult mammalian optic nerve is difficult to regenerate after injury. Transplantation of the peripheral nerve or a Schwann cell (SC) graft can promote injured axonal regrowth. We tried to develop a new type of tissue-engineered SC graft that consisted of SCs seeded onto a poly(lactic-co-glycolic acid)/chitosan conduit. Meanwhile, SCs were transfected along the ciliary neurotrophic factor (CNTF) gene in vitro by electroporation to increase their neurotrophic effect. Four weeks after transplantation, GAP-43 labelled regenerating axons were found in the SC grafts, and axons in the CNTF-SC graft were longer than those in the SC graft. Tissue-engineered SC grafts can provide a feasible environment for optic nerve regeneration and may become an alternative for bridging damaged nerves and repairing nerve defects in the future.     

Enhanced in vivo survival of Schwann cells by a synthetic oxygen carrier promotes sciatic nerve regeneration and functional recovery

Local hypoxia in the early stages of peripheral nerve injury is a challenge for axonal regeneration. To address this issue, perfluorotributylamine (PFTBA)‐based oxygen carrying fibrin hydrogel was prepared and injected into Schwann cell (SC)‐seeded collagen‐chitosan conduits to increase oxygen supply to SCs within the conduits. The conduit containing PFTBA‐SC gel was then applied to bridge a 15‐mm sciatic nerve defect in rats. It was observed that most of the GFP‐labeled SCs initially seeded in the PFTBA hydrogel remained alive for approximately 28 days after their in vivo implantation. The number of SCs was significantly higher in the PFTBA‐SC scaffold than that in the SC scaffold without PFTBA. In addition, nerve regeneration and functional recovery were examined after nerve injury repair. We found that the PFTBA‐SC scaffold was capable of promoting axonal regeneration and remyelination of the regenerated axons. Further studies showed the PFTBA‐SC scaffold was able to accelerate the recovery of motor and sensory function of the regenerating nerves. Electrophysiological analysis showed area under the curve of compound muscle action potential and nerve conduction velocity were also improved, and gastrocnemius muscle atrophy was partially reversed by PFTBA‐SC scaffold. Furthermore, microvessel density analysis showed PFTBA‐SC composites were beneficial for microvascular growth, which provided sustained oxygen for regenerating nerve in the later stages of nerve regeneration. In conclusion, enhanced survival of SCs by PFTBA is capable of promoting sciatic nerve regeneration and functional recovery, which provides a new avenue for achieving better functional recovery in the treatment of peripheral nerve injuries. Copyright © 2016 John Wiley & Sons, Ltd.     

应用MRI和电镜观察pSVPoMact微基因修饰雪氏细胞移植对损伤脊髓的修复作用

Objective To approach the effect of microgene pSVPoMcat modified Schwann cell (SC) on the regeneration and repair of injured spinal cord.Method Spinal cord hemi－ transection models were made with the cutting method in healthy SD rats. Microgene pSVPoMcat modified SC(group A),highly purified SC(group B),and glutin sponge (control group C)were randomly implanted into the cut. After 3 month living ,the host rats were scanned by MRI, and observed under EM. Result Spinal signals at the injury region nearly recovered to normal in group A.No recovery was found in group B.Malacosis was found in group C.TEM findings: regeneration of large number of myelinated and nonmyelinated axons and SC proliferation in group A, myelinated axon regeneration and SC necrosis in group B, non myelinated and nonmyelinated axon in group C.Conclusion Implantation of microgene pSVPoMcat modified SC could promote the repair of injured spinal cord.     

多种神经组织修复成鼠脊髓损伤的实验研究

背景脊髓损伤后以游离周围神经片断(雪旺氏细胞)与胚胎脊髓联合移植能协同诱发宿主再生潜能,而带血管蒂周围神经移植后有更多的雪旺氏细胞存活,但其与胚胎脊髓协同作用的研究较少.目的探讨不同神经组织移植修复成鼠急性脊髓损伤的能力.设计单盲随机对照实验.地点和对象实验在解放军第三军医大学大坪医院全军战创伤中心骨创伤科完成,研究对象为150只4月龄雌性Wistar属成年大鼠,由解放军第三军医大学实验动物中心提供.干预成鼠胸髓损伤后,分别移植游离正中神经(FPN组)、带血管蒂正中神经(VPN组)、孕14天胚胎脊髓(FSC组)、游离正中神经加胚胎脊髓(P+F组)、带血管蒂正中神经加胚胎脊髓(V+F组).主要观察指标术后8周行神经解剖及电生理检查.结果V+F组再生轴突和存活雪旺氏细胞数目、胚胎脊髓体积增长速度和神经元密度显著高于对照组(P＜0.01),细胞分化较好,突触较成熟o SEP的P1,N1波潜伏期显著缩短(P＜0.01).结论带血管蒂周围神经与胚胎脊髓联合移植,在解剖和电生理上均优于其他组织,对FSC的生长发育及损伤神经元的再生能力均有一定的促进作用.     

一种新的生物材料-海藻酸盐修复长距离周围神经缺损的实验研究

目的探讨一种新的神经再生引导材料--可生物降解并吸收的海藻酸盐对兔坐骨神经缺损修复的作用.方法切除兔一侧坐骨神经的一部分,形成一35mm缺损,用两片海藻酸盐制成的海棉状材料,不经缝合地植入缺损并桥接两侧断端,术后行电生理、解剖学、组织学检查.结果术后6周起右下肢体出现电生理上的恢复,术后16周解剖学检查见原缺损处再生组织连接两断端,组织学检查证实再生组织内有大量有髓及无髓的轴突组织,未发现有海藻酸盐的残留.(而在未使用海藻酸盐修复的对照组动物,各项检查均为阴性.)结论海藻酸盐能促进长距离周围神经缺损的再生,同时也表明不缝合修复神经缺损的可能性,这为临床应用提供了新的材料和方法.     

周围神经趋化性再生的细胞和分子生物学机制

周围神经损伤的治疗是临床上的难题。以往研究主要集中在神经损伤后断端缺损的桥接修复和提高神经的再生速度，但实际上功能的恢复更取决于再生轴索能否精确地长入原先支配的靶器官。因此，充分理解神经趋化性再生的细胞和分子生物学基础，在治疗中运用神经再生趋化性理论，对促进神经功能恢复具有重要的意义。本文就周围神经再生趋化性理论及其细胞和分子生物学机制的研究进展作一综述。     

Conductive conduit small gap tubulization for peripheral nerve repair

Despite advances in surgical techniques, functional recovery following epineurial neurorrhaphy of transected peripheral nerves often remains quite unsatisfactory. Small gap tubulisation is a promising approach that has shown potential to traditional epineurial neurorrhaphy in the treatment of peripheral nerve injury. Thus, the goal of this study is to evaluate sciatic nerve regeneration after nerve transection, followed by small gap tubulization using a reduced graphene oxide-based conductive conduit. In vitro, the electrically conductive conduit could promote Schwann cell proliferation through PI3K/Akt signaling pathway activation. In vivo, the results of electrophysiological and walking track analysis suggest that the electrically conductive conduit could promote sensory and motor nerve regeneration and functional recovery, which is based on the mechanisms of selective regeneration and multiple-bud regeneration. These promising results illustrate electrically conductive conduit small gap tubulization as an alternative approach for transected peripheral nerve repair.

rGO-based conductive nerve conduit as a scaffold to bridge peripheral nerve transected injury and 2 mm gap provides a suitable microenvironment for axons selective regeneration.     

Low‐intensity pulsed ultrasound combination with induced pluripotent stem cells‐derived neural crest stem cells and growth differentiation factor 5 promotes sciatic nerve regeneration and functional recovery

The treatment of lengthy peripheral nerve defect is challenging in the field of nerve regeneration. Our previous studies have shown that low‐intensity pulsed ultrasound (LIPUS) could promote the proliferation, cell viability, and neural differentiation of induced pluripotent stem cells‐derived neural crest stem cells (iPSCs‐NCSCs) and improve the regeneration of damaged peripheral nerve. In this study, the mechanical signal transduction pathway of LIPUS promoting iPSCs‐NCSCs proliferation and differentiation was further explored, and the effects of LIPUS combined with iPSCs‐NCSCs, perfluorotributylamine (PFTBA), and growth differentiation factor 5 (GDF5) on the repair of peripheral nerve injury were evaluated. Results showed LIPUS may regulate the proliferation and differentiation of iPSCs‐NCSCs through FAK‐ERK1/2 signal pathway. PFTBA could supply sufficient oxygen to promote the viability of iPSCs‐NCSCs under 5% hypoxia culture condition and provide a favourable microenvironment for nerve regeneration. The addition of GDF5 could promote the neural differentiation of iPSCs‐NCSCs in vitro. LIPUS treatment of allogeneic decellularized nerve conduit containing iPSCs‐NCSCs, PFTBA, and GDF5 has very good effect on the repair of sciatic nerve injury. Taken together, these results provide functional evidence that LIPUS might be a useful tool to explore alternative approaches in the field of nerve regeneration.     

痉挛性脑瘫周围神经的超微结构研究

探讨痉挛性脑瘫周围神经的病理改变特点,取材２５例痉挛性脑瘫患者的脊神经前根及外周末梢神经标本４０个,通过透射电镜观察其亚显微结构改变,结果：脱髓鞘病变范围广,Ｓｃｈｗａｎｎ氏细菌病变先于并严重于轴索,轴索病变相对不明显;未见神经再生迹象,有髓纤维病变明显于无髓纤维,束膜内血管周有淋巴细胞浸润。提示：痉挛性脑瘫的周围神经呈原发性脱髓鞘表现,神经纤维再生能力差,可见免疫反应介入征象。     

人胚雪旺细胞组织工程神经修复坐骨神经缺损的实验研究

目的：探索人胚雪旺细胞作为组织工程的种子细胞修复周围神经缺损的可行性。方法：通过组织工程方法用PLGA导管和polyglactin 910纤维负载人胚雪旺细胞预先构置好人工神经，然后用于修复大鼠20mm的坐骨神经缺损，并与神经切断后原位缝合以及用单纯的PLGA导管进行修复的实验组进行对照。通过活体肢体功能观察、靶器官肌肉测量、电生理检测、辣根过氧化物酶示踪、连续组织切片图像分析以及透射电镜等检查神经再生情况。结果：人工神经修复组神经再生良好，效果接近于神经原位缝合组，明显优于单纯的PLGA导管修复组。结论：人胚雪旺细胞构建的人工神经可以修复20mm的周围神经缺损。