【作者】 邓许勇；

【导师】 金大地；

【作者基本信息】 南方医科大学 ， 骨外科学， 2010， 博士

【摘要】 脊髓损伤(spinal cord injuries, SCI)是现在交通、工矿事故、运动意外以及战伤中常见的损伤,伤者多为青壮年,损伤造成的截瘫不仅严重影响患者的身心健康,而且给家庭和社会造成巨大的经济、人力和精神负担。长期以来脊髓损伤的修复一直是困扰人类健康的难题,也是世界各国科学家们致力于研究的焦点,但目前缺少切实可行的有效治疗方法。脊髓损伤的治疗一直是神经科学研究的难点和热点,传统观点认为中枢神经系统损伤后,由于神经元自身再生能力差和外在微环境的抑制,损伤神经是不可再生的。1981年神经学家在基础研究中证实中枢神经损伤后的神经结构修复是可能的,掀起了各国学者对脊髓修复研究的又一次热潮。SCI后轴突再生障碍的原因相当复杂,其主要原因有：(1)对脊髓的直接损伤及继发炎症使脊髓内功能神经元大量坏死或凋亡且神经元再生困难；(2)脊髓损伤后暴露的髓鞘相关抑制分子、轴突生长抑制性蛋白的大量分泌和释放及继发形成的胶质瘢痕阻碍了轴突的生长和正确连接；(3)损伤造成局部细胞凋亡,致使细胞分泌的神经营养因子减少,破坏了神经元修复和支持轴突再生的有利微环境。目前SCI的修复研究主要集中在以下几个方面：(1)药物治疗：如传统的大剂量皮质类固醇激素(甲基强的松龙)的冲击治疗,可以有效的减轻脊髓的继发性炎症损伤,保护并防止残存的损伤神经元进一步凋亡。目前研究的氯化锂(LiCl)、免疫抑制剂FK506等除了可以保护损伤神经元外,还可以有效的促进损伤轴突再生。(2)细胞移植：细胞移植是目前研究的热点,其原理是利用某些种子细胞可以分泌多种神经营养、黏附和趋化因子,营养并保护损伤神经元、诱导轴突再生及再髓鞘化来修复SCI。既往的研究已为SCI修复提供了较成熟的种子细胞,如：胚胎干细胞(ESCs)、雪旺氏细胞(SCs)、嗅鞘细胞(OECs)、神经干细胞(NSCs)、骨髓间充质干细胞(BMSCs)、脐血干细胞(UCBCs)等,大量研究证实细胞移植均能够在一定程度上促进脊髓神经功能的恢复。(3)组织移植：随着组织工程学的飞速发展,一种更先进的SCI修复理念逐步形成,即修复SCI必须包括组织水平上的重建,整合SCI治疗研究的各种有效策略,在诱导轴突定向再生的同时,减少局部胶质瘢痕形成。目前,应用于脊髓的组织工程支架主要有两种,一种为人工合成可降解高分子生物材料,如：聚乳酸、聚乙醇酸及其共聚物等,另一种为自体或异体组织移植,如异体胚胎脊髓组织、自体肌纤维支架、游离周围神经或带血管蒂的游离周围神经组织移植修复脊髓损伤,也取得了一定的修复效果。(4)改善脊髓损伤局部微环境：SCI后轴突再生困难的另一关键因素就是适宜轴突再生的微环境遭到破坏,各种不利于轴突再生的髓磷脂源性蛋白(myelin, MAG, Nogo-A, Omgp)轴突再生抑制分子大量分泌,而各种神经营养因子(BDNF、GDNF、NGF、NT3等)的缺乏及胶质瘢痕形成阻碍了轴突再生。(5)联合治疗：采取联合不同作用机制的治疗方法,更好的发挥协同作用来促进SCI后神经修复。本研究拟用氯化锂联合脐血干细胞及脊髓去细胞支架移植修复大鼠脊髓全横断损伤,观察该方法对SCI后神经再生和脊髓修复的影响,并探讨其促进脊髓修复的作用机制。目的1.探讨脐血间充质干细胞(MSCs)体外分离、纯化、扩增的方法与条件,以及向神经细胞定向诱导分化的可行性,为脐血干细胞移植治疗脊髓损伤提供理论依据。2.探讨氯化锂体外诱导人脐血间充质干细胞(MSCs)向神经细胞分化的可行性。3.制备脊髓去细胞支架,观测脊髓内基质骨架的结构特点。4.探索氯化锂联合脐血干细胞及脊髓去细胞支架移植对大鼠脊髓全横断损伤的效果及其机制。方法1.无菌条件下收集正常足月儿的脐带血,肝素抗凝,密度梯度离心法分离人脐血单个核细胞,贴壁法纯化,用含15%FBS的低糖DMEM培养基进行扩增培养,流式细胞仪检测表面抗原。20ng/ml bFGF诱导MSCs向神经细胞分化,免疫组化鉴定神经元特异性标志蛋白MAP-2。2.取3代的脐血MSCs分3组进行体外诱导,A组：用含15%FBS和20ng/mlbFGF的DMEM完全培养基预诱导24 h,3mol/LLicl的DMEM培养基继续诱导6d；B组：含3mol/L Licl的DMEM培养基诱导7d；C组：含15% FBS的DMEM培养基正常培养7d。光镜下观察细胞形态,用免疫组化技术检测细胞NSE、MAP-2及GFAP的表达。3.取健康成年SD大鼠脊髓数段,每段长约2cm,手术显微镜去除脊髓表面的脂肪组织和硬脊膜,分为A、B、C三个组,每组取1个标本作为正常对照,其余段均行化学萃取。A、B、C三个组的萃取振荡频率分别为80r/min、120r/min和160r/min。将标本采用Triton X-100和脱氧胆酸钠进行萃取,蒸馏水漂洗后行切片HE染色和扫描电镜观察。萃取后的脊髓置于4℃无菌PBS溶液(0.01mol/L,pH7.4)中保存备用。4.首先经大鼠胸T9节段制备脊髓全横断损伤模型。120只成年雌性SD大鼠随机分为6组,每组20只：①A组为对照组：仅行T9平面脊髓全横断；②B组为Licl组：脊髓全横断+Licl(腹腔注射85mg/kg,1/日)；③C组为细胞移植组：脊髓全横断+UCB-SCs移植；④D组细胞支架联合移植组：脊髓全横断+支架复合hUCB-SCs移植；⑤E组为Licl+细胞移植组：脊髓全横断+hUCB-SCs移植+Licl；⑥F组为Licl+细胞支架联合移植组：脊髓全横断+支架复合hUCB-SCs移植+Licl。于术后1d、3d及每周的最后一天,应用BBB评分评价大鼠脊髓功能的恢复情况；8周后取材,通过HE染色及Brdu细胞核标记观察移植细胞的存活、迁移；通过免疫组化染色的方法,观察hUCB-SCs向神经细胞分化及表达神经纤维的情况；通过荧光金逆行追踪,观察脊髓神经纤维的再生与分布；综合全面评价治疗措施修复脊髓损伤的效果。结果1.采用密度梯度离心法成功从脐血中分离出脐血间充质干细胞,流式细胞仪检测显示细胞不表达或弱表达CD14、CD34和CD45等造血干细胞和内皮细胞标志,但显著表达神经细胞表面标志CD90和间充质干细胞表面标志CD29、CD105。经20ng/ml bFGF诱导14天后长梭形的MSCs胞体收缩,呈圆形、不规则多边形,细胞出现类似神经元形态。兔疫组化染色MAP-2呈弱阳性表达。2.A组与B组诱导3d后细胞即出现形态学上的改变,细胞变成不规则形,立体感增强,从胞体伸出突起。免疫组织化学和免疫荧光方法鉴定显示,诱导后的细胞能表达神经元特异性标志NSE和MAP-2,阳性表达率A组(分别为73.6±7.8%,75.5±8.5%)高于B组(分别为31.0±4.3%,33.5±5.0%)(P<0.05),而星形胶质细胞特异性标志GFAP阳性细胞较少,A、B、C三组阳性表达率分别为4.7±3.3%、5.1±4.6%、8.5±3.2%。3.采用化学萃取方法可以成功制备大鼠脊髓去细胞支架,其扫描电镜结果显示,采用120r/min的震荡频率萃取的脊髓去细胞支架内基质纤维结构保存较好,基质纤维走形与脊髓纵轴一致,呈波浪状平行排列,彼此之间有短横向的基质纤维相互连接成三维镂空的网状结构。4.术后8周C、D、E、F组可观察至Brdu标记的脐血干细胞在体内存活并在脊髓内迁移,其中D、F组细胞存活数量多于C、E组(P<0.05)。FITC荧光标记NF-200阳性神经纤维的表达,在A组和B组未见表达；D、F组阳性表达数高于C、E组(P<0.05),可见少量连续性神经纤维通过损伤区。荧光金逆行脊髓追踪显示E组和F组在脊髓损伤区头侧中有少量被荧光金标记的神经锥体细胞,而C组和D组偶见被荧光金标记的神经锥体细胞,对照组和单纯氯化锂组未见被荧光金标记的神经锥体细胞。后肢功能运动BBB评分除对照组和单纯氯化锂组间无统计学差异外,E组和F组的BBB评分较其他组提高(P<0.05),其中,F组好于E组,D组好于C组。结论1.脐血中可以分离并扩增MSCs；脐血MSCs经bFGF体外诱导可以向神经元样细胞分化,并部分表达神经元特异性标志MAP-2。2.Licl体外可诱导人脐血MSCs分化为神经元样细胞,结合生长因子bFGF预诱导可大大提高其诱导效果。3.采用化学萃取方法可以成功制备大鼠脊髓去细胞支架；脊髓去细胞支架保存有天然的脊髓内基质纤维骨架,可有效的引导神经细胞和神经纤维定向生长和迁移。4.氯化锂能促进人脐血间充质干细胞在损伤区的存活并向神经细胞分化,脊髓去细胞支架内天然的脊髓基质骨架能为脊髓两侧残端基质提供良好对接,阻止外源性瘢痕长入,引导神经细胞和神经轴突定向生长和迁移,氯化锂联合脐血干细胞与脊髓去细胞支架移植能够促进细胞移植修复大鼠脊髓损伤的效果。更多还原

【Abstract】 Spinal cord injury(SCI) is a common injury in transport, mining accidents, sports injuries and warfare nowadays. Most of the injured are young people. The paraplegia caused by SCI not only seriously affects the physical and mental health of patients, but also put up to families and the community tremendous economic, human and spiritual burden. For a long time the repair of spinal cord injury has been plaguing human health problems, and the world scientists are working on the focus, but still lack of practical and effective therapy methods. Curing spinal cord injury is always a difficult and hot point in neuroscience research. It’s believed that normal mature mammalian central nervons system couldn’t regenerate after injured, because of poor regeneration capability of neuron and the inhibitor form glial microenvironment. Since 1981 the neuroscientist confirmed that the repair of the central nervous system structure after injury was possible, the study on repair of SCI has been becoming hot spot in many academic institute. The reasons that interfering axon regeneration after SCI were extremely complicated. First, primary and secondary injury on spinal cord caused a great of spinal cord functional neurons apoptosis and necrosis, which making the regeneration of axons difficulty. Second, a great quantity secretion and release of myelin-associated inhibitor and axon growth inhibition protein after SCI. The secondary glial scar impeded the axons growth and properly connected. Third, the decreased secretion of many neurotrophic factors resulting from partial cells apoptosis destroyed the micro-environment benefiting for neurons repair and axon regeneration. The measures of repairing SCI currently are mainly the following aspects:drug treatment, cell transplantation, tissue transplantation, improve the local micro-environment and union treatment. The traditional high-dose corticosteroids(methylprednisolone) impacting treatment could effectively reduce the secondary inflammation after SCI, and protect the residual damaging neurons and prevent the further apoptosis. At present, the studies claimed that lithium chloride(Licl)and the immunosuppressant FK506, and so on, could protect neurons from injury and could effectively promote axon regeneration. Cell transplantation is currently a hot research, the principle is to use some seed cells secreting a variety of neurotrophic, adhesion agents, nutrite and protect the neuron after injury, and then induced axon regeneration and re-myelinization to repair SCI. Previous studies have provided a more mature seeds cells for SCI, such as: embryonic stem cells(ESCs), schwann cells(SCs), olfactory ensheathing cells (OECs), neural stem cells NSCs), bone marrow mesenchymal stem cells(BMSCs), the umbilical cord blood stem cells(UCBCs). A large number of studies have confirmed that cell transplantation facilitates the restoration of spinal cord function. With the tissue engineering rapid development, a more advanced concept of SCI repairment appeared gradually that repair of SCI must include the organization levels reconstruction. Integrating effective treatments of SCI repair strategy, we could reduce glial scar formation at the same time induce the directional axon regeneration. At present, there were two engineering scaffolds applicating in spinal cord tissue, one was biodegradable polymer synthetic biological material, such as polylactic acid, and polyglycolic acid copolymer, another was autologous or allogeneic tissue transplantation such as allogeneic embryonic spinal cord tissue, autologous muscle fiber frame, free of peripheral nerve(FPN), or the free vascularized peripheral nerve(VPN) tissue transplantation to repair spinal cord injury, and achieved a certain degree of effect. Another key factor for axon regenerate obstacle after SCI is the appropriate axonal regeneration micro-environment has been damaged, and myelin-derived protein(myelin,MAG, Nogo-A, Omgp) which inhibited the axon regeneration were greatly secreted, and lacking of various neurotrophic factor (BDNF and GDNF, NGF, NT3)and the glial scar formation hindered axon regeneration. To this end, a new therapy strategy Combined with several methods in different mechanism was used, which may have a better synergy to promote nerve repair after SCI. Accordingly,this study was designed to apply lithium chloride combined with transplantation of human umbilical cord blood mesenchymal stem cells and acellular spinal cord scaffold to repair SCI. From this study, the nerve regeneration and function recovery of SCI animal will be investigated, at the same time, the possible mechanism of recovery in SCI will be discussed.Obsjective:1. To investigate the method and conditions of isolation, proliferation of multipotent mesenchymal stem cells(MSCs)from human umbilical cord blood in vitro, and he possibility of inducing human umbilical cord blood mesenchymal stem cells (MSCs) to differentiate into neuron-like cells.2. To investigate the effects and possible mechanisms of treating complete transected spinal cord in rats through lithium chloride combined with transplantation of human umbilical cord blood mesenchymal stem cells and acellular spinal cord scaffold3. To explore a method for fabricating the acellular spinal cord scaffold and to observe the construction features of the scaffold.4. To investigate the possibility of inducing human umbilical cord blood mesenchymal stem cells (MSCs) to differentiate into neuron-like cells by lithium chloride (LiCl) in vitro.Methods:1. Human umbilical cord blood was collected from mature neonates. All samples were obtained sterilely with 20 U/ml heparin. The cord mononuclear cells were isolated with lymphocyte separation medium (density 1.077g/ml), then purified by wall sticking screening and expanded with slight sugar DMEM containing 15%FBS. Immunophenotypes of the cells surface were analyzed by flow eytometry. Expanded MSCs were induced to differentiate into neuron-like cells in medium added with basic fibroblast growth factor(bFGF), and immonohistochemical staining was used to identify the specific protein of neuron:microtubule associated protein-2(MAP-2)。2. The third passage of the expanded MSCs were pre-inducted with DMEM containing 15%FBS and 20ng/ml bFGF for 24 hours, then induced with DMEM without serum but 3mol/L Licl for 6 days in group A. The MSCs were induced with DMEM containing 3mol/L Licl for 7 days in group B. The MSCs were normally cultured with DMEM containing 15%FBS in group C. The morphological changes of the cells were observed under phase contrast microscope. The neuron specific markers containing neuron specific enolase(NSE), microtubule associated protein-2(MAP2) and glial fibrillary acid protein(GFAP) were evaluated by indirect immunocyto-chemistry staining.3. Several Segments of spinal cord were obtained from Adult female Sprague-Dawley rats. Under operative microscopy, the fat and a part of dural matter were cutted before the extraction procedure. Segments of spinal cord obtained from rats were divided randomly into three groups: group A(frequency of vibration=80r/min), group B(frequency of vibration=120r/min) and group C (frequency of vibration=160r/min). The spinal cord Was delt with solution of Triton X-100 and with solution of sodium deoxycholate at room temperature. Then washed with distilled-water, delt with HE staining and observed under light microscope. Scanning electron microscope was used to observe the ultramicrostructure. The fabricated acellular spinal cord scaffolds were stored in 0.01mol/L PBS(PH7.4).4. At first, to establish the entire transected spinal cord injury model at T9 level in rats. Then 120 Sprague-Dawley (SD) female rats were divided randomly into six groups,20 in each group. Group A(spinal cord entire transaction), group B (spinal cord entire transaction+intraperitoneal injection of 85mg/kg lithium chloride every day), group C (spinal cord entire transaction+hUCB-SCs transplantation), group D (spinal cord entire transaction+hUCB-SCs transplantation+intraperitoneal injection lithium chloride), group E (spinal cord entire transaction+acellular spinal cord scaffold and hUCB-SCs transplantation), group F (spinal cord entire transaction+ acellular spinal cord scaffold and hUCB-SCs transplantation+intraperitoneal injection lithium chloride). At 1 day,3day and the end day of every week post-operation, a behavioral testing was performed weekly upon each hindlimb of all animals according to the BBB scoring system. At the 8th week, all animals were sacrificed and the spinal cords were taken out for morphological observation. Tissues in SCI sites were observed with H&E staining and Brdu nuclear labeling to identify the survival and migration of SCs. With immunocyto-chemistry staining to identify the differentiation of neuron-like cells and expression of the neurofibras. With fluorescent-gold(FG) spinal cord retrograde tracing to observe the regeneration and distribution of spinal nerve fiber. The treatment effects of spinal cord injury were identified comprehensively.Results:1. The hUCB-SCs could be isolated successfully from the UCB with density gradient centrifugation. Flow cytometry analysis showed that the cells were positive for CD90 (neural cell antigen)and CD29, CD105 (MSC-specific surface markers), while negative for CD34 (hematopoietic stem cell antigen)and CD45 (leukocyte common antigen). MSCs differentiated into neuron-like cells induced for 14 days by 20ng/ml bFGF and the differentiated cells faintly expressed MAP-2.2. After inducted for 3 days, morphological changes were observed obviously in group A and B.6 days later, the differentiated cells showed typical neuronal morphology. The expression of NSE and MAP2 were positive for the majority cells in group A and B, but that of group A [(73.6±7.8)%, (75.5±8.5)%]were obviously higher than group B [(31.0±4.3)%, (3.5±5.0)%]. few expressed GFAP in both groups.3. Acellular spinal cord scaffold can be fabricated by chemical extraction. The three-dimensional(3D)structure of the acellular spinal cord scaffold are kept intact, in which there are bundles of extracellular matrix (ECM) fiber which aligned lengthways were interlaced with transversal matrix fibers.4. It could be observed that Brdu marked hUCB-SCs survivaed and migrated in the spinal cord postoperative 8th week in groupC, D, E and F. The survivaed hUCB-SCs in group D and F were extremely more than that in group C and E. Through FITC fluorescence labeling NF-200 positive nerve fibers, No NF-200 positive nerve fibers could be observed in group Aand B; NF-200 positive nerve fibers in group D and F were extremely more than that in group C and E. Some continuited nerve fibers through injury district could be observed in group D and F. Fluorogold(FG)retrograde tracing show that a small amount of pyramidal cells were labeled by FG in group E and F. The nerve pyramidalcells marked by FG occasionally were observed in group C and D, the remaining two groups were not apperence. There were significant differences of BBB Score of hindlimb functional movement among all groups except group Aand B. The BBB Score of hindlimb functional movement were better in group F than that in group E, better in group D than that in group C.Conclusions:1. The results suggest that MSCs can be obtained from HUCB. MSCs from HUCB can be induced to differentiate into neuron-like cells and faintly express MAP-2 in vitro.2. Licl could induce the human umbilical cord blood MSCs to differentiate into neuron-like cells in vitro. Licl combined with bFGF could improve the induced effects.3. Acellular spinal cord scaffold can be fabricated by chemical extraction. The native 3D structure of the ECM in the scaffold maybe provide the structural foundation for inducing effectively the neurons and axons to directionally grow and migrate.4. Licl could improve survival and differentiation into neural cells of the human umbilical cord blood in injury district. Acellular spinal cord scaffold could bridge the both stumps of the injured spinal cord by means of "alete butt joint", stop the in-migrating of the peripheral tissues around the spinal cord, guide the directional growth and migration of the neural cells and axons. Licl combined with transplantation of human umbilical cord blood mesenchymal stem cells and acellular spinal cord scaffold could improve the functional recovery of the hindlimbs in the complete transected spinal cord rats.更多还原