嗅鞘细胞复合PLGA导管修复周围神经缺损的研究

探讨嗅鞘细胞(OECs)复合聚乳酸-聚羟基乙酸共聚物(PLGA)导管对大鼠坐骨神经缺损的修复作用。方法:SD大鼠80只,随机分成4组,切除右侧部分神经干造成10mm的神经缺损。OECs PLGA组用充满细胞外基质凝胶和OECs悬液(CM-DiI预标记)的PLGA导管桥接坐骨神经缺损;OECs 硅胶管组用含相同内容物的硅胶管桥接;PLGA组和硅胶管组则分别用充满细胞外基质凝胶和DMEM/F12培养基的PLGA导管和硅胶管桥接。术后每周进行感觉运动功能检测,8周时行腓肠肌湿重恢复率、乙酰胆碱脂酶(AChE)染色、电生理和组织形态学分析等检测,同时移植细胞的两组每周进行细胞示踪观察。结果:移植细胞沿神经纵轴分布;除坐骨神经功能指数(SFI)指标外,OECs PLGA组的各项再生功能指标均优于其它三组。结论:OECs复合PLGA导管能够促进再生神经的成熟和靶组织功能的恢复,二者联合移植是一种有效的周围神经缺损修复方法。     

三维取向PLGA神经导管促进坐骨神经再生的实验研究

目的:探讨应用改进静电纺丝技术一次成型制备三维(3D)取向聚乳酸与聚羟基乙酸共聚物(PLGA)纳米神经导管的可行性,检测其对坐骨神经再生的促进作用。方法:应用改进的静电纺丝技术制备无缝取向PLGA纳米神经导管,通过扫描电镜和透射电镜检测支架的纳米结构;分别制备取向和非取向纳米纤维支架修复13mm坐骨神经缺损模型。36只成年SD大鼠随机分为3组(每组12只),A组:非取向PLGA神经导管组(阴性对照);B组:取向PLGA神经导管组,C组:自体神经移植组(阳性对照),于术后3月通过大体观察、行走足印分析、腓肠肌萎缩率、电生理检测、组织形态学检测、透射电镜检测及图像分析,评价无缝取向PLGA纳米神经导管修复坐骨神经缺损的效果。结果:神经导管修复神经缺损三月后,大体观察显示神经导管结构完整,无坍塌和断裂;各组再生神经均有通过神经导管长入远端。B组与C组的腓肠肌萎缩率和神经电传导速度无统计学差异(P0.05),均优于A组。B组与C组再生神经纤维数量及成熟程度均要明显优于A组。结论:无缝取向PLGA纳米神经导管能够诱导并促进神经再生,提高坐骨神经再生的质量,有望成为自体神经移植的替代物。     

三维取向PLGA神经导管促进坐骨神经再生的实验研究

目的：探讨应用改进静电纺丝技术一次成型制备三维（3D）取向聚乳酸与聚羟基乙酸共聚物（PLGA）纳米神经导管的可行性,检测其对坐骨神经再生的促进作用。方法：应用改进的静电纺丝技术制备无缝取向PLGA纳米神经导管,通过扫描电镜和透射电镜检测支架的纳米结构;分别制备取向和非取向纳米纤维支架修复13mm坐骨神经缺损模型。36只成年SD大鼠随机分为3组（每组12只）,A组：非取向PLGA神经导管组（阴性对照）;B组：取向PLGA神经导管组,C组：自体神经移植组（阳性对照）,于术后3月通过大体观察、行走足印分析、腓肠肌萎缩率、电生理检测、组织形态学检测、透射电镜检测及图像分析,评价无缝取向PLGA纳米神经导管修复坐骨神经缺损的效果。结果：神经导管修复神经缺损三月后,大体观察显示神经导管结构完整,无坍塌和断裂;各组再生神经均有通过神经导管长入远端。B组与C组的腓肠肌萎缩率和神经电传导速度无统计学差异（P〈0.05）,均优于A组。B组与C组再生神经纤维数量及成熟程度均要明显优于A组。结论：无缝取向PLGA纳米神经导管能够诱导并促进神经再生,提高坐骨神经再生的质量,有望成为自体神经移植的替代物。     

植入血管束的血管化人工神经导管对受损神经功能恢复影响的实验研究

目的:研究植入血管束的血管化人工神经导管修复SD大鼠长段坐骨神经缺损对神经功能恢复的影响。方法:将18只成年雌性SD大鼠制成14mm的大鼠坐骨神经缺损模型后,随机分为3组(每组12条神经),分别采用不同的修复方法。A组:自体神经移植组(自体组);B组:普通PGLA神经导管移植组(导管组);C组:植入自体血管束的普通PGLA神经导管移植组(血管化导管组)。观察术后大鼠后肢皮肤溃疡面积;检测术后6周、12周时步态变化和肌电图。结果:术后各组SD大鼠均出现后肢溃疡,血管化导管组SD大鼠后肢溃疡愈合较导管组早2周。血管化导管组步态检测SFI明显优于导管组,与自体神经移植组无明显差异。肌电图检测表明血管化导管组无论是神经传导速度,还是动作电位振幅均明显大于导管组(P<0.05),与自体神经移植组无明显差异(P>0.05)。结论:植入血管束的血管化人工神经导管能有效地促进受损神经的功能恢复。     

聚己内酯/壳聚糖神经导管复合骨髓间充质干细胞促进大鼠坐骨神经损伤修复的研究

目的:观察聚己内酯/壳聚糖神经导管复合骨髓间充质干细胞修复大鼠坐骨神经缺损的效果。方法:将24只SD大鼠随机分为4组,制备右侧坐骨神经5mm缺损模型,A组聚己内酯/壳聚糖神经导管复合骨髓间充质干细胞移植组;B组聚己内酯神经导管复合骨髓间充质干细胞移植组;C组壳聚糖神经导管复合骨髓间充质干细胞移植组;D组自体神经移植组。术后每2周进行坐骨神经功能指数检测,12周时行电生理、腓肠肌湿重恢复率、组织学观察和免疫组织化学检测。结果:坐骨神经功能指数显示,A组运动功能恢复速度较B、C组快,但比D组慢。A组电生理和腓肠肌湿重恢复率的检测结果与C、D组相比无统计学意义(P0.05),但优于B组(P0.05)。组织学观察,A组再生神经纤维排列密集。S-100免疫组织化学结果表明A组有大量雪旺细胞增生。结论:聚己内酯/壳聚糖神经导管复合骨髓间充质干细胞能够促进周围神经损伤修复,效果与壳聚糖神经导管、自体神经相同,优于聚己内酯神经导管。     

周围神经损伤后外源性GDNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型 ,局部给予胶质细胞源性神经营养因子 (GDNF) ,应用尼氏染色、酶组织化学染色方法 ,观察到外源性GDNF能减少脊髓修复侧前角运动神经元死亡的数目 ,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶 (CHE)及酸性磷酸酶 (ACP)变化的幅度。这表明外源性GDNF能保护周围神经切断后引起的神经元损伤。     

转基因细胞片复合PLGA支架修复兔软骨缺损的研究

细胞片技术是应用组织工程方法使培养细胞从培养表面分离而形成含有细胞外基质的一层完整片状结构,弥补了传统组织工程技术的不足,是获取种子细胞以及对种子细胞进行转移的一项新技术。为探讨体外生长分化因子-5(GDF5)基因转染修饰的BMSCs细胞片与GDF5转基因BMSCs负载的PLGA支架形成的共聚物修复兔甲状软骨缺损的效果,实验通过腺病毒转染GDF5基因至四代兔BMSCs,温度敏感性培养皿制备GDF5转基因细胞片并与负载有转染GDF5基因BMSCs的PLGA支架复合,移植至同种兔甲状软骨缺损处,分别于术后4、8周行大体观察和组织学检测其修复效果。实验分3组:(A)转基因细胞片包裹负载有转基因BMSCs的PLGA支架组;(B)负载有转基因BMSCs的PLGA支架组;(C)负载BMSCs的PLGA支架组。结果显示,体外成功收获了完整的GDF5转基因细胞片,Real time PCR检测到GDF5 mRNA的表达,行大体组织的II型胶原免疫组化和阿利新蓝染色显示:A组和B组均表达II型胶原和糖胺聚糖(GAG),但A组表达高于B组,有统计学意义(P0.05)。由此可得,转基因细胞片包裹负载转基因BMSCs PLGA支架较传统转基因BMSCs负载PLGA支架方法具有更加优越的成软骨能力,能更有效地促进软骨缺损的修复。     

担载神经生长因子(NGF)的乳液法涂层纤维体外缓释研究

目的:目前周围神经修复中,神经导管是研究热点,本文研究乳液法涂层纤维制备的神经导管在神经修复中应用的可能性。方法:本文采用乳液法制备担载NGF的丝素-聚乳酸(PLLA)涂层电纺纤维,观察纤维的形态,测定NGF的体外释放动力学参数,并考察纤维释放液对于PC12细胞增殖的影响。结果:担载NGF的涂层纤维具备类似于细胞外基质(ECM)的三维结构和多孔形态;涂层纤维中NGF体外有效缓释10天;细胞实验中,在含有释放液的培养基中生长的PC12细胞,与空白对照组相比,荧光强度平均多了2000-4000个荧光强度,所以释放液可以更好地促进PC12细胞的增殖。结论:担载NGF的乳液法涂层纺丝纤维具备促进缺损周围神经修复的条件,可以进一步研究在动物体内修复缺损周围神经中的效果,为以后的临床应用打下基础。     

乳鼠坐骨神经植块法的雪旺氏细胞培养

目的:改善并建立一种新的大鼠雪旺氏细胞(SCs)的培养方法,为研究外周神经损伤修复模型及其它外周神经相关实验提供高纯度、多数量的SCs。方法:麻醉后显微镜下解剖并分离新生3天内SD大鼠的坐骨神经,采取植块培养的方法,显微镜下尽量剥除坐骨神经纤维外膜,并梳理松解坐骨神经的神经纤维束。梳理后剪碎坐骨神经,每小块种植于培养皿中,使用纯血清培养4小时,再加入正常的DMEM/F12培养基,消化培养2-3代。最后用S-100及GFAP免疫荧光染色进行纯度鉴定。结果:本实验在总结前人实验的基础上,联合创新采用坐骨神经外膜剥除、神经内膜梳理、纯血清培养以及胰酶差速消化等方法,短时间内获得SCs的纯度可达99%以上,可用于进一步对雪旺氏细胞的功能进行研究。结论:这种选用乳鼠坐骨神经植块、血清培养的方法简单易操作,无需额外的生长因子及抑制因子,可在短期内获得大量高纯度的SCs。     

骨髓间充质干细胞与施万细胞联合移植对大鼠周围神经 损伤端侧吻合的修复效果研究

目的:探究骨髓间充质干细胞(MSCs)与施万细胞(SCs)联合移植对大鼠周围神经损伤端侧吻合的修复效果。方法:选取SD雌性大鼠60只均制作成坐骨神经损伤端侧吻合模型,并将其随机分为联合移植组、MSCs组和SCs组,分别对吻合端进行骨髓间充质干细胞与SCs联合移植、MSCs移植、SCs移植。观察分析三组大鼠的神经电生理学指标和腓神经功能指数(PFI)和神经传导速度(NCV)。结果:三组大鼠的PFI和NCV均有所改善,且联合移植组的PFI和NCV均优于其他两组,并随着时间推移损伤坐骨神经功能恢复越来越好。结论:MSCs与SCs均具有促进大鼠周围神经身上修复的功能,且两种细胞联合移植效果更加明显。     

Engineering a prokaryotic apocytochrome c as an efficient substrate for Saccharomyces cerevisiae cytochrome c heme lyase

In spite of the extensive research using induced pluripotent stem (iPS) cells, the therapeutic potential of iPS cells in the treatment of peripheral nerve injury is largely unknown. In this study, we repaired peripheral nerve gaps in mice using tissue-engineered bioabsorbable nerve conduits coated with iPS cell-derived neurospheres. The secondary neurospheres derived from mouse iPS cells were suspended in each conduit (4000,000 cells per conduit) and cultured in the conduit in three-dimensional (3D) culture for 14 days. We then implanted them in the mouse sciatic nerve gaps (5 mm) (iPS group; n=10). The nerve conduit alone was implanted in the control group (n=10). After 4, 8 and 12 weeks, motor and sensory functional recovery in mice were significantly better in the iPS group. At 12 weeks, all the nerve conduits remained structurally stable without any collapse and histological analysis indicated axonal regeneration in the nerve conduits of both groups. However, the iPS group showed significantly more vigorous axonal regeneration. The bioabsorbable nerve conduits created by 3D-culture of iPS cell-derived neurospheres promoted regeneration of peripheral nerves and functional recovery in vivo. The combination of iPS cell technology and bioabsorbable nerve conduits shows potential as a future tool for the treatment of peripheral nerve defects.     

成年大鼠雪旺细胞的快速扩增

采用接种雪旺细胞的可降解导管修复外周神经损伤是一种有望替代自体神经移植的方法。如何在短期内利用病人少量的神经碎片获得大量雪旺细胞是该方法用于临床的关键。以大鼠坐骨神经为模型,利用雪旺细胞增殖的内在机制,探索出一种快速增殖成年雪旺细胞的方法。采用预变性7d的坐骨神经,用酶消化分离出雪旺细胞,接种在层粘连蛋白包被的培养瓶中,经过7d的培养,获得纯度为96％、细胞密度为600个／mm^2的雪旺细胞,雪旺细胞的纯度和密度明显高于对照的新鲜神经。未使用霍乱毒素、毛喉素等促有丝分裂剂和抑制成纤维细胞的基因毒素,符合临床使用要求。结果表明,可以利用少量的损伤神经碎片在短期内获得大量可用于临床的雪旺细胞。     

Peripheral Nerve Injuries Treatment: a Systematic Review

Patients with peripheral nerve injuries, especially severe injury, often face poor nerve regeneration and incompletely functional recovery, even after surgical nerve repair. Current researches have extensively focused on the new approaches for the treatment of peripheral nerve injuries. This review summarizes treatments of peripheral nerve injures, from conventional suturing method, to conduit coaptation with stem cell and growth factor, and review the developments of research and clinical application of these therapies.     

Sciatic nerve regeneration by transplantation of menstrual blood-derived stem cells

This is the first study demonstrating the efficacy of menstrual blood-derived stem cell (MenSC) transplantation via a neural guidance conduit, for peripheral nerve regeneration. The synthesized poly (?-caprolactone)/Gelatin conduit, filled with collagen type I and seeded with 3?×?10⁴ MenSCs, was implanted into a rat’s 10 mm sciatic nerve defect. The results of hot plate latency, sciatic functional index and weight-loss percentage of wet gastrocnemius muscle demonstrated that the MenSC transplantation had comparable nerve regeneration outcome to autograft, as the gold standard of nerve bridging. The transplantation of MenSCs via a synthetic conduit could ameliorate the functional recovery of sciatic nerve-injured rats which make them a potential candidate for cell therapy of peripheral nervous system disorders.     

Magnetic resonance imaging monitoring dual-labeled stem cells for treatment of mouse nerve injury

Background aimsAdipose-derived stem cells (ADSCs) have shown great promise in the regenerative repair of injured peripheral nerves. Magnetic resonance imaging (MRI) has provided attractive advantages in tracking superparamagnetic iron oxide nanoparticle (SPION)-labeled cells and evaluating their fate after cell transplantation. This study investigated the feasibility of the use of MRI to noninvasively track ADSCs repair of peripheral nerve injury in vivo.MethodsGreen fluorescent protein (GFP)-expressing ADSCs were isolated, expanded, differentiated into an SC-like phenotype (GFP-dADSCs) at early passages and subsequently labeled with SPIONs. The morphological and functional properties of the GFP-dADSCs were assessed through the use of immunohistochemistry. The intracellular stability, proliferation and viability of the labeled cells were evaluated in vitro. Through the use of a microsurgical procedure, the labeled cells were then seeded into sciatic nerve conduits in C57/BL6 mice to repair a 1-cm sciatic nerve gap. A clinical 3-T MRI was performed to investigate the GFP-dADSCs in vitro and the transplanted GFP-dADSCs inside the sciatic nerve conduits in vivo.ResultsThe GFP-dADSCs were efficiently labeled with SPIONs, without affecting their viability and proliferation. The labeled cells implanted into the mice sciatic nerve conduit exhibited a significant increase in axonal regeneration compared with the empty conduit and could be detected by MRI. Fluorescent microscopic examination, histological analysis and immunohistochemistry confirmed the axon regeneration and MRI results.ConclusionsThese data will elucidate the neuroplasticity of ADSCs and provide a new protocol for in vivo tracking of stem cells that are seeded to repair injured peripheral nerves.     

Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat

Quantification of peripheral nerve regeneration in animal studies of nerve injury and repair by histologic, morphologic, and electrophysiologic parameters has been controversial because such studies may not necessarily correlate with actual nerve function. This study modifies the previously described sciatic functional index (SFI), tibial functional index (TFI), and peroneal functional index (PFI) based on multiple linear regression analysis of factors derived from measurements of walking tracks in rats with defined nerve injuries. The factors that contributed to these formulas were print-length factor (PLF), toe-spread factor (TSF), and intermediary toe-spread factor (ITF). It was shown that animals with selective nerve injuries gave walking tracks that were consistent, predictable, and based on known neuromuscular deficits. The new formula for sciatic functional index was compared with previously described indices. The sciatic functional index, tibial functional index, and peroneal functional index offer the peripheral nerve investigator a noninvasive quantitative assessment of hindlimb motor function in the rat with selective hindlimb nerve injury.