脊髓脱细胞支架复合人脐血间充质干细胞促进大鼠脊髓长节段缺损轴突长入再生及功能恢复
|
摘要
研究背景：
脊髓是中枢神经系统的重要组成部分,脊髓损伤(Spinal cord injury, SCI)后几乎无再生能力,导致肢体运动、感觉功能丧失,甚至死亡；SCI对患者生活及社会影响巨大,近年干细胞及组织工程(Tissue Engineering, TE)治疗SCI取得一些进展是目前一个研究热点,但仍然没有有效的治疗方法满足临床需要。通常,自然无干预情况下,损伤脊髓经历了原发损伤和继发损伤的病理过程：①脊髓神经元死亡,凋亡和缺乏修复能力；②促进修复的因素如神经营养因子缺乏,微环境的变化；③中枢神经生长抑制因子的存在；④引导系统退化导致轴突不能沿正确方向生长：⑤损伤部位增殖的成纤维细胞、星型胶质细胞、小胶质细胞和上皮细胞形成神经胶质瘢痕,阻碍脊髓再生。而原发损伤细胞裂解释放的毒素又可导致继发损伤平面上下两侧的脊髓组织。
鉴于SCI病理的复杂性,治疗策略应包括多个因素,联合应用多种治疗措施,不仅保护残存的神经元存活,而且能调节损伤脊髓的微环境,促进轴突再生,重建神经反射环路。SCI目前的治疗原理包括：①补充神经营养因子促进轴突存活与再生；②中和中枢神经生长抑制因子；③减少受损细胞变性和损失；④细胞移植补充受损细胞；⑤减少瘢痕和空洞,提供轴突生长的基质。到目前为止,SCI的动物实验尚没有满足临床治疗效果的有效对策。其中主要原因之一是由于传统的自体移植、同种异体移植或不同的合成支架移植都存在多方面的缺陷,例如：细胞来源受限、细胞功能改造、存在伦理学问题、免疫排斥反应、更重要的是人工支架不能模仿靶组织中细胞外基质(Extracellular matrix, ECM)三维结构等。更何况再生神经轴突通过支架时,容易出现弥散性生长而迷失生长方向。如何才能克服这些神经再生的不利因素呢?利用TE技术(即应用细胞、支架和生长因子,以多元联合移植的方式修复和重建受损组织)可能可以帮助损伤脊髓恢复功能,重建结构。近年的基础和临床研究也证实TE技术可以有效地帮助组织功能恢复及结构重建。以种子细胞复合生物支架移植为代表的脊髓修复研究的进展成为有潜力的SCI修复策略。
其中,干细胞复合支架移植是其中研究的热点,干细胞是具有自我复制和多向分化潜能的原始细胞,是形成生命机体各种组织器官的起源细胞。在不同微环境中能够自我更新及分化或横向分化为不同类型衍生细胞,可以用来替代病变或衰老的细胞。通常,干细胞分为胚胎干细胞(Embryonic stem cell, ESC)和成体干细胞。后者可来自骨髓、外周血、脐血(Cord blood, CB)等,它们具有神经细胞分化潜能并能分泌生长因子,促使受损神经轴突再生通过脊髓内空洞损伤区,并为之提供细胞迁移所必须的有益微环境或替代受损细胞。其中具有潜力的细胞之一脐血间充质干细胞(Cord blood mesenchymal stem cells, CB-MSCs)相比较其他来源的间充质干细胞(Mesenchymal stem cells, MS Cs)具有以下明显的优点：①来源丰富,易于获得；②采集方便,对供者无任何痛苦；③CB中免疫系统处于原始阶段,移植后急、慢性移植物抗宿主反应(Graft-versus-host reaction, GVHR)发生率及严重程度均轻；④CB中存在较原始的造血干细胞、MSCs以及内皮干／祖细胞等,具有更强的增殖、分化能力；⑤CB中各种病毒感染机会相对少。
支架是TE技术另一个主要方面,理想的支架应具有多方面有益特性：仿生三维空间结构和彼此连通的孔隙网络,与靶组织相类似的生物力学性能,良好的生物学相容性以及对细胞因子传输功能等；以促进和引导细胞与组织的再生。近期才有报道的脱细胞脊髓(Acellular Spinal Cord, ASC)支架具备高度仿生特点,相比各种人工支架更复合脊髓组织特点。有潜力作为脊髓修复支架：桥接脊髓两侧残端、促进宿主神经细胞再生、抑制胶质瘫痕形成；能引导植入种子细胞的增殖、定向分化和迁移；引导神经轴突定向延伸、以重建脊髓神经环路、最终促进脊髓神经功能的修复。
尚没有关于高度仿生的ASC支架体内修复SCI的报道。我们这项研究利用仿生的大鼠ASC支架复合人脐血间充质干细胞(Human umbilical cord blood-derived mesenchymal stem cells, hUCB-MSCs)异种移植修复大鼠SCI缺损,观察疗效,并通过组织学,凋亡,局部免疫状况探讨机制,为将来SCI临床治疗提供基础和依据。
目的：
1.制备大鼠支架,观测脊髓内基质骨架的结构特点；体外分离人脐血间充质干细胞及扩增鉴定。
2.探索大鼠脱细胞脊髓支架复合人脐血间充质干细胞促进脊髓损伤长节段轴突长入再生及功能恢复的效果,探讨其机制。
3.探索大鼠脱细胞脊髓支架复合人脐血间充质干细胞修复脊髓半切缺损相关局部免疫调节及凋亡研究,从凋亡及局部免疫状况角度探讨相关机制。
第一部分大鼠脊髓脱细胞支架促进大鼠半切脊髓缺损功能恢复
目的：评价通过冻融萃取法获得高度仿生的大鼠ASC支架是否具有促进大鼠SCI运动功能恢复,以及其促进SCI功能恢复与宿主自体神经细胞迁移粘附,增殖分化的关系。
方法：通过冻融萃取法获得高度仿生的ASC支架,观察大体,组织结构及超微结构；随机选取12只SD大鼠制作脊髓右半切缺损模型,随机分配到两个实验组：A组,SCI组(n=6)；B组,SCI+ASC支架组(n=6)。术后每周运用Basso, Beattie, and Bresnahan locomotor test (BBB)评分进行运动功能评价。分别于术后2周及8周进行标本神经细胞免疫荧光染色(Immunofluorescencestaining,IFS)和髓鞘碱性蛋白(Myelin basic protein,MBP)染色。两组术后右下肢功能学BBB评分采用SPSS20.0统计软件进行统计学分析,比较两组计量分析行Students't检验,检验水准α=0.05,P<0.05表示有统计学差异。
结果：ASC支架大体观呈乳白色,缺乏韧性,柔软；横截面为ECM构成的网络结构,充满空隙,表现为均一,平行排列的淡红色纤维结构；大多数的细胞,髓鞘和轴突从ASC支架上去除；截面具有三维网络结构；HE(Hematoxylin and eosin)染色观察：SCI组,显示脊髓右半切缺损模型完全破坏毁损脊髓背侧和腹侧右侧部分；SCI+ASC支架移植组：大鼠ASC支架非常好的整合入大鼠脊髓半切缺损,可见大量细胞核分布在ASC支架上；大鼠右后肢运动功能学：SCI+ASC支架移植组显著促进大鼠脊髓损伤后运动功能的恢复(P<0.05):IFS观察：几乎没有星型胶质细胞可以在支架上观察到；有少量神经元细胞和神经干细胞分布在支架上；而ASC支架植入物的主要分布神经细胞类型是少突胶质细胞,另外,MBP免疫组化示术后8周观察到髓鞘化轴突长入支架。
结论：大鼠脱细胞脊髓支架是非常具有潜力修复脊髓损伤的材料,该支架通过改造局部微环境促进宿主少突胶质细胞增殖并促进轴突再髓鞘化。
第二部分脊髓脱细胞支架复合人脐血间充质干细胞促进脊髓损伤长节段轴突长入再生及功能恢复
目的：近年来通过干细胞治疗SCI取得了一些进展。而最近,具有脊髓修复潜力高度仿生天然脊髓组织ECM的ASC支架已经成功制备。本研究旨在探讨脊髓半切缺损损伤的是否可以通过植入高度仿生的ASC支架,及ASC支架复合hUCB-MSCs种子细胞获得功能改善及相关神经组织细胞形态学变化。
方法：建立成年SD大鼠(n=36)SCI右半切损伤缺损模型,术后各组(A组：SCI+ASC支架、(n=12)；B组,SCI+ASC+hUCB-MSCs、(n=12)；C组：SCI、(n=12))分别立即植入仿生植入物。术后在每周各组进行大鼠行为学BBB评分测试,共8周。术后第二周及第八周进行神经细胞标志物免疫荧光检测,并在第八周完成生物素葡聚糖胺(biotinylated dextran amine,BDA)示踪切片检查。采用单因素方差分析(One-way ANOVA)检验各组大鼠行为学BBB评分统计差异,方差齐性后各组两两比较采用Bonferroni's进行比较,以a=0.05为检验标准,P<0.05表示有统计学差异。
结果：第三代hUCB-MSCs流式细胞仪(Flow cytometry,FCM)鉴定结果：该细胞低表达CD14(0.31%).CD34(0.82%).CD45(0.23%).HLA-DR(0.26%),并高表达以下粘附分子CD90(99.99%).CD29(92.95%).CD44(99.98%).CD105(99.92%)和CD73(99.88%),鉴定结果与MSCs的免疫表型相关报道一致。hUCB-MSCs复合ASC支架体外培养两天后大体观白色,柔软；5-溴脱氧尿苷(5-bromodeoxyuridine, BRDU)标记阳性的hUCB-MSCs分布在ASC支架ECM构成的三维网络结构内。术后大鼠行为学BBB评分各植入物组相比无植入组具有显著的运动功能改善(P<0.01)。行为学BBB评分两植入组(A组：SCI+ASC支架；B组,SCI+ASC+hUCB-MSCs)之间除第三周(P<0.05)外,其余个时间点无显著性差异(P>0.05)。BRDU标记阳性的hUCB-MSCs在植入后2周尚可观察到,但未观察发现其向神经细胞分化。此外,两植入组(A组：SCI+ASC支架；B组,SCI+ASC+hUCB-MSCs)植入物内见大量宿主神经细胞(主要是少突胶质细胞)。BDA追踪试验表明,有再生的宿主轴突生长入植入物,作为一个直接证据证明轴突再生的效果。扫描电镜(Scanning electron microscopy,SEM)观察两移植物组(A组：SCI+ASC支架；B组,SCI+ASC+hUCB-MSCs)移植物内存在较厚的髓鞘及有髓神经纤维。
结论：我们的研究首次提供了实验证据表明,大鼠脱细胞脊髓支架与人脐血间充质干细胞能够促进大鼠脊髓损伤轴突再生和功能恢复。
第三部分大鼠脊髓脱细胞支架复合人脐血间充质干细胞修复脊髓半切缺损相关局部免疫调节及凋亡研究
目的：制作大鼠脊髓半切模型,观察ASC支架及其联合hUCB-MSCs移植修复SCI早期,对宿主免疫细胞损伤脊髓局部分布及凋亡的影响,探讨移植物恢复脊髓功能的机制。
方法：按照前两部分法制作ASC支架,及分离培养hUCB-MSCs:取成年SD大鼠36只,随机分组：A组：SCI(n=12)；B组：SCI+ASC支架(n=12)；C组：SCI+ASC+hUCB-MSCs(n=12)；大鼠行脊髓半切缺损模型后分别植入相应组别植入物,于术后两周每组随机各取6只动物取材做免疫细胞标志物IFS及Tunel阳性细胞检测,高倍镜下计数阳性细胞数量；另每组各取6只动物取材行caspase-3活化度检测,计数OD(Optical Density)值。采用单因素方差分析(One-way ANOVA)检验各组阳性细胞数及caspase-3活化OD值的统计差异,方差齐性后各组两两比较采用Bonferroni's进行比较,以a=0.05为检验标准,P<0.05表示有统计学差异。
结果：C组(SCI+ASC+hUCB-MSCs)早期术后两周,明显抑制Tunel阳性细胞数(P<0.05)及caSpase3活化度(P<0.05),抑制凋亡；而B组(SCI+ASC)并无抑制凋亡作用。C组(SCI+ASC+hUCB-MSCs)植入脊髓损伤后两周明显抑制中性粒细胞(P<0.05)、巨噬细胞(小胶质细胞)(P<0.05)、T淋巴细胞(P<0.05)在脊髓损伤区趋化,增殖；而B组(SCI+ASC)未引发明显的免疫细胞局部分布差异,与A组(SCI)相比较,局部炎症细胞阳性细胞数量无明显差异(P>0.05)。C组(SCI+ASC+hUCB-MSCs)相比较其他两组早期对于1gM阳性表达无明显差异(P>0.05)。
结论：大鼠脱细胞脊髓支架复合人脐血间充质干细胞修复脊髓半切缺损早期通过抑制凋亡,调节抑制局部免疫细胞趋化、增殖、促进脊髓功能恢复。
Background:
The spinal cord is an important part of the central nervous system, and It is almost have no ability to regenerate by itself after spinal cord injury. The most cases of spinal cord injury always result in loss of limb movements and sensory function, or even death. Those patients caused enormous impact of life and society. Stem cells and tissue engineering made some progress in the treatment of spinal cord injury in recent years, and it is a hot research topic. However, there is still no effective treatment to meet clinical needs. Typically, natural non-intervention in the case of spinal cord injury experienced primary injury and secondary injury pathological processes which include:①The death and apoptosis of spinal cord nerve cells, and lack of ability to repair by itself;②The lack of neurotrophic factors and micro-environment change;③The presence of inhibitory factor in Central nervous system;④Boot system degradation causes axons can't be growth in the right direction;⑤Proliferation of fibroblasts, astrocytes, microglia cells and epithelial cells to form glial scar in the Injury site, which hinder spinal cord regeneration. Moreover, the primary damaged cells release toxins and can cause secondary damage on both up and down sides of the spinal cord tissue.
Due to the complexity of the pathology of spinal cord injury, the treatment strategy of spinal cord injury should include a number of factors and variety of treatment measures. It is not only to protect the remnants of nerve neuron survival, but also can regulate the micro-environment of the injured spinal cord and promote axonal regeneration and reconstruction of nerve reflex loop. Now days, the spinal cord injury treatment principles are as follows:①Supple neurotrophic factors to promote axonal survival and regeneration;②Reduce inhibitory factors in the central nervous system;③Reduce cell degeneration and losses;④Cells transplantation to replacement of the damaged cells;⑤Reduce scarring and cavities in favor of the axonal growth. Unfortunately, so far there are no animal studies of spinal cord injury meet the clinical requirements. Because of many defects in the traditional allograft and synthetic scaffolds, Such as:a limited cell sources, cell function transformation, ethical issues and immune rejection, among them the most important is that the artificial scaffold cannot mimic the three-dimensional structure of the extracellular matrix in the target tissue. Not to mention the regeneration of axons through the bracket, it is easy to get lost disseminated growth direction. How can we overcome these unfavorable factors for nerve regeneration? Using tissue engineering technology as the cells, scaffolds, and growth factors may be able to help spinal cord injury recovery and reconstruction. Basic and clinical research in recent years also confirmed that tissue engineering techniques can help restore tissue function and structure reconstruction. The progress of seed cells in biological scaffold graft repair of spinal cord injury has to be potential research strategy in spinal cord injury repair.
Using Stem cell seeded in scaffold as a graft in treatment of SCI is a hot research point. Stem cell has ability of self-replication and differentiation potential, and it's the origin of cells of various tissues and organs in living organisms. Microenvironments determine the capable of self-renewal and differentiation for different types of cells to replace diseased or aging cells. Typically, Stem cells divide into embryonic stem cells, and adult stem cells. The adult stem cells can be derived from the bone marrow, peripheral blood, cord blood, and they have the nerve cell differentiation potential and the secretions of growth factors, prompted impaired axonal regeneration through the spinal cord cavity damage zone, and to whom provide beneficial environment or replacement of damaged cells. The Umbilical cord blood mesenchymal stem cells have the following obvious advantages compared to other sources:①Rich sources and readily available;②The acquisition convenient of donor with no pain;③Cord blood is in the primitive stage of immune system after transplantation, which caused light acute and chronic graft-versus-host response in the incidence and severity;④Cord blood have primitive hematopoietic stem cells, mesenchymal stem cells and endothelial progenitor cells, have a stronger proliferation, differentiation capacity;⑤Umbilical cord blood in a variety of relatively few opportunities for infection.
The ideal scaffold should have many different characteristics:bionic three-dimensional structure and pore network to communicate with each other, have similar biomechanical properties with the target tissue, good biological compatibility and the cytokines transmission function to promote and guide cell and tissue regeneration. Recently It is reported the acellular spinal cord matrix scaffolds have a high degree of bionic characteristics, compared to a variety of artificial scaffold composite spinal cord tissue characteristics. It has Potential as spinal cord repair scaffold which means bridge on both sides of spinal cord, promote the host regeneration of nerve cells and inhibit glial scar formation; It would be able to guide the implanted seed cell proliferation, directed differentiation and migration and guide axons directional extension to rebuild spinal cord loops, and promote the repair of spinal cord function.
The acellular spinal cord scaffolds repair of spinal cord injury in vivo has not reported yet. This study aim to use acellular spinal cord scaffold seeded with xenograft cells of human umbilical cord blood-derived mesenchymal stem cells as the graft to repair spinal cord injury and observe the function recovery of rat were, to explore the mechanism for spinal cord injury by histology, apoptosis and local immune status. this study may provided the basis and foundation for the future clinical treatment.
Objective:
1. Prepared the acellular spinal cord scaffold, observing the characteristics of the skeleton structure of the spinal cord matrix; isolated human umbilical cord blood-derived mesenchymal stem cells in vitro and identification;
2. Explore the acellular spinal cord scaffold seeded with human umbilical cord blood-derived mesenchymal stem cells promote axonal growth regeneration and functional recovery effect of long distance of the spinal cord injury, and explore its mechanism.
3. Explore the mechanisms of the rat spinal cord injury treat with the acellular spinal cord scofflds seeded with umbilical cord blood mesenchymal stem cells from the point of view of apoptosis and local immune status.
Chapter I Regeneration of Spinal cord with Acellular Spinal Cord Scaffolds:An in vivo study in rat model of Hemi-sectioned spinal cord injury
Objective:Acellular spinal cord (ASC) scaffold was prepared through chemical extraction, and in vivo promote spinal cord regeneration on lateral hemisected thoracic spinal cord in adult SD Rats. And to test the effect of ASC scaffold on functional recovery after spinal cord injury in vivo study by supporting adhesion, proliferation of host neural cells and axonal remyelination.
Methods:Twenty-four adult Sprague Dawley (SD) rats were conducted lateral hemisection in thoracic spinal cord to establish SCI model. The ASC scaffolds were implanted into the SCI lesion gaps in ASC group (n=6) while the lesion only as injury only group(n=6). The Basso, Beattie, and Bresnahan (BBB) locomotor test were conducted to assess neurologic function weekly for8weeks.14day and56day after SCI, six rats in each group were sacrificed and measured under histological, immunohistochemical and MBP examination.
Results:The ASC scaffold general looking is milky white, the lack of toughness and softness; The cross-section of it is a network structure of the extracellular matrix constituent, The most of the cells and myeloid have removed. Moreover, The ASC scaffold arranged in parallel fibrous structure and cross-section has a three dimensional network structure. In animal test the ASC scaffold could integrate well with the host spinal cord at the spinal cord gap and have beneficial effect on functional recovery (p<0.05). HE staining showed the group of spinal cord injury destroy the damaged spinal cord dorsal and ventral right part. The ASC scaffold graft group:The ASC scaffold have very good integration into the rat spinal cord hemisection defect and seen a large number of cell nuclei distributed in the ASC scaffold. Immunohistochemical showed few astrocytes was observed on ASC graft, forming an astrocyte-free area and, also, a few neural stem cells and neurons were observed on grafts. Among them, oligodendrocytes is main cell type, distributed in ASC graft at day56post-SCI. Some remyelination of ingrown axons had taken place inside in ASC scaffold.
Conclusion:ASC Scaffold, as a potential scaffold for SCI, could provide a microenvironment favorable to differentiation or proliferation of host oligodendrocyte and promote axon remyelination.
Chapter II Acellular Spinal Cord Scaffold Seeded with Mesenchymal Stem Cells Promotes Long-Distance Axon Regeneration and Functional Recovery in Spinal Cord Injured Rats
Objective:Stem cells based experimental therapies are partially successful for the recovery of Spinal Cord Injury (SCI). Recently, acellular spinal cord (ASC) scaffolds which mimic native extracellular matrix (ECM) have been successfully prepared. This study aimed to investigate whether the spinal cord lesion gap could be bridged by implantation of bionics designed ASC scaffold, either alone or seeded with human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), and their effects on functional improvement.
Methods:A laterally hemisected SCI lesion was performed in adult Sprague-Dawley (SD) rats (n=36) and ASC scaffolds were implanted into the lesion immediately, either with or without seeded hUCB-MSCs. All rats were behaviorally tested using the Basso-Beattie-Bresnahan (BBB) test once a week for8weeks. we detect nerve cells by immunofluorescence at second and eighth week after SCI, and completed BDA tracer at eighth week
Results:The three passgers of umbilical cord blood mesenchymal stem cells identified by flow cytometry:the cells with low expression of CD14(0.31%), CD34(0.82%), CD45(0.23%), HLA-DR (0.26%), and high expression following adhesion molecule CD90(99.99%), CD29(92.95%), CD44(99.98%) CD105(99.92%) and CD73(99.88%), and it is consistent with identification of mesenchymal stem cell phenotype reported before; human umbilical cord blood mesenchymal stem cells seeded in ASC scaffold were cultured in vitro for two days, and gross looking is white, soft and distribution of5-bromodeoxyuridine (Brdu)-positive human umbilical cord blood-derived mesenchymal stem cells in the scaffold of extracellular matrix composed of a three-dimensional network. Behavioral analysis showed that there was significant locomotor recovery improvement in combined treatment group (ASC scaffold and hUCB-MSCs) as compared with the control SCI only group (no scaffold)(p<0.01). Brdu-labeled hUCB-MSCs could also be observed in the implanted ASC two weeks after implantation. Moreover, host neural cells (mainly oligodendrocytes) were able to migrate into the graft. Biotin-dextran-amine (BDA) tracing test demonstrated that myelinated axons successfully grew into the graft and subsequently promoted axonal regeneration at lesion sites.
Conclusion:Our study provides the first experimental evidence that ASC scaffold seeded with hUCB-MSCs is able to bridge a spinal cord cavity and promote long-distance axon regeneration and functional recovery in SCI rats
Chapter Ⅲ The local immune regulation and apoptosis research on Acellular Spinal Cord Scaffold Seeded with Mesenchymal Stem Cells to repair spinal cord hemisection defects
Objective:explore the apoptosis and immune regulation mechanisms of rat spinal cord hemisection model of spinal cord injury repaired by Acellular Spinal Cord Scaffold Seeded with Mesenchymal Stem Cells
Method:Spinal cord acellular scaffold seed with human umbilical cord blood-derived mesenchymal stem cells as graft.36adult SD rats were randomized to three groups:group A, the SCI only group (n=12); group B, SCI+ASC scaffold (n=12); group C, SCI+ASC+hUCB-MSCs (n=12); The rat underwent spinal cord hemisection respectively implanted corresponding group. Two weeks after operation, Three animals of each group detect markers of immune cells and Tunel-positive cells by immunofluorescence and count positive cells; another three animals in each group under caspase-3activation degree detection, counting the OD value. Using analysis of variance to test the statistical differences in each group the number of positive cells and caspase-3activation OD values, differences between groups of homogeneity of variance with LSD test, P<0.05indicates statistical difference.
Results:two weeks after operation the group of SCI+ASC+hUCB-MSCs significantly inhibited Tunel positive cells (P<0.05) and caspase-3activation (P <0.05), it means this group inhibit apoptosis of local neural cells; On the contrary, the ASC scaffold group did not inhibit apoptosis. Moreover, the group of SCI+ASC+hUCB-MSCs significantly inhibited neutrophil (P<0.05), macrophages (microglia)(P<0.05), T lymphocytes (P<0.05) proliferation in spinal cord injury area. While, The ASC scaffold group doesn't significant inhibit immune response, It compared with SCI only group that there is no obvious differences on inflammatory positive cells (P>0.05). Furthermore the three groups was no significant difference on1gM positive expression (P>0.05).
Conclusion:The Acellular Spinal Cord Scaffold Seeded with Mesenchymal Stem Cells to repair spinal cord hemisection defects promote founction recovery through suppression of local immune cells, and inhibit apoptosis.
脊髓是中枢神经系统的重要组成部分,脊髓损伤(Spinal cord injury, SCI)后几乎无再生能力,导致肢体运动、感觉功能丧失,甚至死亡；SCI对患者生活及社会影响巨大,近年干细胞及组织工程(Tissue Engineering, TE)治疗SCI取得一些进展是目前一个研究热点,但仍然没有有效的治疗方法满足临床需要。通常,自然无干预情况下,损伤脊髓经历了原发损伤和继发损伤的病理过程：①脊髓神经元死亡,凋亡和缺乏修复能力；②促进修复的因素如神经营养因子缺乏,微环境的变化；③中枢神经生长抑制因子的存在；④引导系统退化导致轴突不能沿正确方向生长：⑤损伤部位增殖的成纤维细胞、星型胶质细胞、小胶质细胞和上皮细胞形成神经胶质瘢痕,阻碍脊髓再生。而原发损伤细胞裂解释放的毒素又可导致继发损伤平面上下两侧的脊髓组织。
鉴于SCI病理的复杂性,治疗策略应包括多个因素,联合应用多种治疗措施,不仅保护残存的神经元存活,而且能调节损伤脊髓的微环境,促进轴突再生,重建神经反射环路。SCI目前的治疗原理包括：①补充神经营养因子促进轴突存活与再生；②中和中枢神经生长抑制因子；③减少受损细胞变性和损失；④细胞移植补充受损细胞；⑤减少瘢痕和空洞,提供轴突生长的基质。到目前为止,SCI的动物实验尚没有满足临床治疗效果的有效对策。其中主要原因之一是由于传统的自体移植、同种异体移植或不同的合成支架移植都存在多方面的缺陷,例如：细胞来源受限、细胞功能改造、存在伦理学问题、免疫排斥反应、更重要的是人工支架不能模仿靶组织中细胞外基质(Extracellular matrix, ECM)三维结构等。更何况再生神经轴突通过支架时,容易出现弥散性生长而迷失生长方向。如何才能克服这些神经再生的不利因素呢?利用TE技术(即应用细胞、支架和生长因子,以多元联合移植的方式修复和重建受损组织)可能可以帮助损伤脊髓恢复功能,重建结构。近年的基础和临床研究也证实TE技术可以有效地帮助组织功能恢复及结构重建。以种子细胞复合生物支架移植为代表的脊髓修复研究的进展成为有潜力的SCI修复策略。
其中,干细胞复合支架移植是其中研究的热点,干细胞是具有自我复制和多向分化潜能的原始细胞,是形成生命机体各种组织器官的起源细胞。在不同微环境中能够自我更新及分化或横向分化为不同类型衍生细胞,可以用来替代病变或衰老的细胞。通常,干细胞分为胚胎干细胞(Embryonic stem cell, ESC)和成体干细胞。后者可来自骨髓、外周血、脐血(Cord blood, CB)等,它们具有神经细胞分化潜能并能分泌生长因子,促使受损神经轴突再生通过脊髓内空洞损伤区,并为之提供细胞迁移所必须的有益微环境或替代受损细胞。其中具有潜力的细胞之一脐血间充质干细胞(Cord blood mesenchymal stem cells, CB-MSCs)相比较其他来源的间充质干细胞(Mesenchymal stem cells, MS Cs)具有以下明显的优点：①来源丰富,易于获得；②采集方便,对供者无任何痛苦；③CB中免疫系统处于原始阶段,移植后急、慢性移植物抗宿主反应(Graft-versus-host reaction, GVHR)发生率及严重程度均轻；④CB中存在较原始的造血干细胞、MSCs以及内皮干／祖细胞等,具有更强的增殖、分化能力；⑤CB中各种病毒感染机会相对少。
支架是TE技术另一个主要方面,理想的支架应具有多方面有益特性：仿生三维空间结构和彼此连通的孔隙网络,与靶组织相类似的生物力学性能,良好的生物学相容性以及对细胞因子传输功能等；以促进和引导细胞与组织的再生。近期才有报道的脱细胞脊髓(Acellular Spinal Cord, ASC)支架具备高度仿生特点,相比各种人工支架更复合脊髓组织特点。有潜力作为脊髓修复支架：桥接脊髓两侧残端、促进宿主神经细胞再生、抑制胶质瘫痕形成；能引导植入种子细胞的增殖、定向分化和迁移；引导神经轴突定向延伸、以重建脊髓神经环路、最终促进脊髓神经功能的修复。
尚没有关于高度仿生的ASC支架体内修复SCI的报道。我们这项研究利用仿生的大鼠ASC支架复合人脐血间充质干细胞(Human umbilical cord blood-derived mesenchymal stem cells, hUCB-MSCs)异种移植修复大鼠SCI缺损,观察疗效,并通过组织学,凋亡,局部免疫状况探讨机制,为将来SCI临床治疗提供基础和依据。
目的：
1.制备大鼠支架,观测脊髓内基质骨架的结构特点；体外分离人脐血间充质干细胞及扩增鉴定。
2.探索大鼠脱细胞脊髓支架复合人脐血间充质干细胞促进脊髓损伤长节段轴突长入再生及功能恢复的效果,探讨其机制。
3.探索大鼠脱细胞脊髓支架复合人脐血间充质干细胞修复脊髓半切缺损相关局部免疫调节及凋亡研究,从凋亡及局部免疫状况角度探讨相关机制。
第一部分大鼠脊髓脱细胞支架促进大鼠半切脊髓缺损功能恢复
目的：评价通过冻融萃取法获得高度仿生的大鼠ASC支架是否具有促进大鼠SCI运动功能恢复,以及其促进SCI功能恢复与宿主自体神经细胞迁移粘附,增殖分化的关系。
方法：通过冻融萃取法获得高度仿生的ASC支架,观察大体,组织结构及超微结构；随机选取12只SD大鼠制作脊髓右半切缺损模型,随机分配到两个实验组：A组,SCI组(n=6)；B组,SCI+ASC支架组(n=6)。术后每周运用Basso, Beattie, and Bresnahan locomotor test (BBB)评分进行运动功能评价。分别于术后2周及8周进行标本神经细胞免疫荧光染色(Immunofluorescencestaining,IFS)和髓鞘碱性蛋白(Myelin basic protein,MBP)染色。两组术后右下肢功能学BBB评分采用SPSS20.0统计软件进行统计学分析,比较两组计量分析行Students't检验,检验水准α=0.05,P<0.05表示有统计学差异。
结果：ASC支架大体观呈乳白色,缺乏韧性,柔软；横截面为ECM构成的网络结构,充满空隙,表现为均一,平行排列的淡红色纤维结构；大多数的细胞,髓鞘和轴突从ASC支架上去除；截面具有三维网络结构；HE(Hematoxylin and eosin)染色观察：SCI组,显示脊髓右半切缺损模型完全破坏毁损脊髓背侧和腹侧右侧部分；SCI+ASC支架移植组：大鼠ASC支架非常好的整合入大鼠脊髓半切缺损,可见大量细胞核分布在ASC支架上；大鼠右后肢运动功能学：SCI+ASC支架移植组显著促进大鼠脊髓损伤后运动功能的恢复(P<0.05):IFS观察：几乎没有星型胶质细胞可以在支架上观察到；有少量神经元细胞和神经干细胞分布在支架上；而ASC支架植入物的主要分布神经细胞类型是少突胶质细胞,另外,MBP免疫组化示术后8周观察到髓鞘化轴突长入支架。
结论：大鼠脱细胞脊髓支架是非常具有潜力修复脊髓损伤的材料,该支架通过改造局部微环境促进宿主少突胶质细胞增殖并促进轴突再髓鞘化。
第二部分脊髓脱细胞支架复合人脐血间充质干细胞促进脊髓损伤长节段轴突长入再生及功能恢复
目的：近年来通过干细胞治疗SCI取得了一些进展。而最近,具有脊髓修复潜力高度仿生天然脊髓组织ECM的ASC支架已经成功制备。本研究旨在探讨脊髓半切缺损损伤的是否可以通过植入高度仿生的ASC支架,及ASC支架复合hUCB-MSCs种子细胞获得功能改善及相关神经组织细胞形态学变化。
方法：建立成年SD大鼠(n=36)SCI右半切损伤缺损模型,术后各组(A组：SCI+ASC支架、(n=12)；B组,SCI+ASC+hUCB-MSCs、(n=12)；C组：SCI、(n=12))分别立即植入仿生植入物。术后在每周各组进行大鼠行为学BBB评分测试,共8周。术后第二周及第八周进行神经细胞标志物免疫荧光检测,并在第八周完成生物素葡聚糖胺(biotinylated dextran amine,BDA)示踪切片检查。采用单因素方差分析(One-way ANOVA)检验各组大鼠行为学BBB评分统计差异,方差齐性后各组两两比较采用Bonferroni's进行比较,以a=0.05为检验标准,P<0.05表示有统计学差异。
结果：第三代hUCB-MSCs流式细胞仪(Flow cytometry,FCM)鉴定结果：该细胞低表达CD14(0.31%).CD34(0.82%).CD45(0.23%).HLA-DR(0.26%),并高表达以下粘附分子CD90(99.99%).CD29(92.95%).CD44(99.98%).CD105(99.92%)和CD73(99.88%),鉴定结果与MSCs的免疫表型相关报道一致。hUCB-MSCs复合ASC支架体外培养两天后大体观白色,柔软；5-溴脱氧尿苷(5-bromodeoxyuridine, BRDU)标记阳性的hUCB-MSCs分布在ASC支架ECM构成的三维网络结构内。术后大鼠行为学BBB评分各植入物组相比无植入组具有显著的运动功能改善(P<0.01)。行为学BBB评分两植入组(A组：SCI+ASC支架；B组,SCI+ASC+hUCB-MSCs)之间除第三周(P<0.05)外,其余个时间点无显著性差异(P>0.05)。BRDU标记阳性的hUCB-MSCs在植入后2周尚可观察到,但未观察发现其向神经细胞分化。此外,两植入组(A组：SCI+ASC支架；B组,SCI+ASC+hUCB-MSCs)植入物内见大量宿主神经细胞(主要是少突胶质细胞)。BDA追踪试验表明,有再生的宿主轴突生长入植入物,作为一个直接证据证明轴突再生的效果。扫描电镜(Scanning electron microscopy,SEM)观察两移植物组(A组：SCI+ASC支架；B组,SCI+ASC+hUCB-MSCs)移植物内存在较厚的髓鞘及有髓神经纤维。
结论：我们的研究首次提供了实验证据表明,大鼠脱细胞脊髓支架与人脐血间充质干细胞能够促进大鼠脊髓损伤轴突再生和功能恢复。
第三部分大鼠脊髓脱细胞支架复合人脐血间充质干细胞修复脊髓半切缺损相关局部免疫调节及凋亡研究
目的：制作大鼠脊髓半切模型,观察ASC支架及其联合hUCB-MSCs移植修复SCI早期,对宿主免疫细胞损伤脊髓局部分布及凋亡的影响,探讨移植物恢复脊髓功能的机制。
方法：按照前两部分法制作ASC支架,及分离培养hUCB-MSCs:取成年SD大鼠36只,随机分组：A组：SCI(n=12)；B组：SCI+ASC支架(n=12)；C组：SCI+ASC+hUCB-MSCs(n=12)；大鼠行脊髓半切缺损模型后分别植入相应组别植入物,于术后两周每组随机各取6只动物取材做免疫细胞标志物IFS及Tunel阳性细胞检测,高倍镜下计数阳性细胞数量；另每组各取6只动物取材行caspase-3活化度检测,计数OD(Optical Density)值。采用单因素方差分析(One-way ANOVA)检验各组阳性细胞数及caspase-3活化OD值的统计差异,方差齐性后各组两两比较采用Bonferroni's进行比较,以a=0.05为检验标准,P<0.05表示有统计学差异。
结果：C组(SCI+ASC+hUCB-MSCs)早期术后两周,明显抑制Tunel阳性细胞数(P<0.05)及caSpase3活化度(P<0.05),抑制凋亡；而B组(SCI+ASC)并无抑制凋亡作用。C组(SCI+ASC+hUCB-MSCs)植入脊髓损伤后两周明显抑制中性粒细胞(P<0.05)、巨噬细胞(小胶质细胞)(P<0.05)、T淋巴细胞(P<0.05)在脊髓损伤区趋化,增殖；而B组(SCI+ASC)未引发明显的免疫细胞局部分布差异,与A组(SCI)相比较,局部炎症细胞阳性细胞数量无明显差异(P>0.05)。C组(SCI+ASC+hUCB-MSCs)相比较其他两组早期对于1gM阳性表达无明显差异(P>0.05)。
结论：大鼠脱细胞脊髓支架复合人脐血间充质干细胞修复脊髓半切缺损早期通过抑制凋亡,调节抑制局部免疫细胞趋化、增殖、促进脊髓功能恢复。
Background:
The spinal cord is an important part of the central nervous system, and It is almost have no ability to regenerate by itself after spinal cord injury. The most cases of spinal cord injury always result in loss of limb movements and sensory function, or even death. Those patients caused enormous impact of life and society. Stem cells and tissue engineering made some progress in the treatment of spinal cord injury in recent years, and it is a hot research topic. However, there is still no effective treatment to meet clinical needs. Typically, natural non-intervention in the case of spinal cord injury experienced primary injury and secondary injury pathological processes which include:①The death and apoptosis of spinal cord nerve cells, and lack of ability to repair by itself;②The lack of neurotrophic factors and micro-environment change;③The presence of inhibitory factor in Central nervous system;④Boot system degradation causes axons can't be growth in the right direction;⑤Proliferation of fibroblasts, astrocytes, microglia cells and epithelial cells to form glial scar in the Injury site, which hinder spinal cord regeneration. Moreover, the primary damaged cells release toxins and can cause secondary damage on both up and down sides of the spinal cord tissue.
Due to the complexity of the pathology of spinal cord injury, the treatment strategy of spinal cord injury should include a number of factors and variety of treatment measures. It is not only to protect the remnants of nerve neuron survival, but also can regulate the micro-environment of the injured spinal cord and promote axonal regeneration and reconstruction of nerve reflex loop. Now days, the spinal cord injury treatment principles are as follows:①Supple neurotrophic factors to promote axonal survival and regeneration;②Reduce inhibitory factors in the central nervous system;③Reduce cell degeneration and losses;④Cells transplantation to replacement of the damaged cells;⑤Reduce scarring and cavities in favor of the axonal growth. Unfortunately, so far there are no animal studies of spinal cord injury meet the clinical requirements. Because of many defects in the traditional allograft and synthetic scaffolds, Such as:a limited cell sources, cell function transformation, ethical issues and immune rejection, among them the most important is that the artificial scaffold cannot mimic the three-dimensional structure of the extracellular matrix in the target tissue. Not to mention the regeneration of axons through the bracket, it is easy to get lost disseminated growth direction. How can we overcome these unfavorable factors for nerve regeneration? Using tissue engineering technology as the cells, scaffolds, and growth factors may be able to help spinal cord injury recovery and reconstruction. Basic and clinical research in recent years also confirmed that tissue engineering techniques can help restore tissue function and structure reconstruction. The progress of seed cells in biological scaffold graft repair of spinal cord injury has to be potential research strategy in spinal cord injury repair.
Using Stem cell seeded in scaffold as a graft in treatment of SCI is a hot research point. Stem cell has ability of self-replication and differentiation potential, and it's the origin of cells of various tissues and organs in living organisms. Microenvironments determine the capable of self-renewal and differentiation for different types of cells to replace diseased or aging cells. Typically, Stem cells divide into embryonic stem cells, and adult stem cells. The adult stem cells can be derived from the bone marrow, peripheral blood, cord blood, and they have the nerve cell differentiation potential and the secretions of growth factors, prompted impaired axonal regeneration through the spinal cord cavity damage zone, and to whom provide beneficial environment or replacement of damaged cells. The Umbilical cord blood mesenchymal stem cells have the following obvious advantages compared to other sources:①Rich sources and readily available;②The acquisition convenient of donor with no pain;③Cord blood is in the primitive stage of immune system after transplantation, which caused light acute and chronic graft-versus-host response in the incidence and severity;④Cord blood have primitive hematopoietic stem cells, mesenchymal stem cells and endothelial progenitor cells, have a stronger proliferation, differentiation capacity;⑤Umbilical cord blood in a variety of relatively few opportunities for infection.
The ideal scaffold should have many different characteristics:bionic three-dimensional structure and pore network to communicate with each other, have similar biomechanical properties with the target tissue, good biological compatibility and the cytokines transmission function to promote and guide cell and tissue regeneration. Recently It is reported the acellular spinal cord matrix scaffolds have a high degree of bionic characteristics, compared to a variety of artificial scaffold composite spinal cord tissue characteristics. It has Potential as spinal cord repair scaffold which means bridge on both sides of spinal cord, promote the host regeneration of nerve cells and inhibit glial scar formation; It would be able to guide the implanted seed cell proliferation, directed differentiation and migration and guide axons directional extension to rebuild spinal cord loops, and promote the repair of spinal cord function.
The acellular spinal cord scaffolds repair of spinal cord injury in vivo has not reported yet. This study aim to use acellular spinal cord scaffold seeded with xenograft cells of human umbilical cord blood-derived mesenchymal stem cells as the graft to repair spinal cord injury and observe the function recovery of rat were, to explore the mechanism for spinal cord injury by histology, apoptosis and local immune status. this study may provided the basis and foundation for the future clinical treatment.
Objective:
1. Prepared the acellular spinal cord scaffold, observing the characteristics of the skeleton structure of the spinal cord matrix; isolated human umbilical cord blood-derived mesenchymal stem cells in vitro and identification;
2. Explore the acellular spinal cord scaffold seeded with human umbilical cord blood-derived mesenchymal stem cells promote axonal growth regeneration and functional recovery effect of long distance of the spinal cord injury, and explore its mechanism.
3. Explore the mechanisms of the rat spinal cord injury treat with the acellular spinal cord scofflds seeded with umbilical cord blood mesenchymal stem cells from the point of view of apoptosis and local immune status.
Chapter I Regeneration of Spinal cord with Acellular Spinal Cord Scaffolds:An in vivo study in rat model of Hemi-sectioned spinal cord injury
Objective:Acellular spinal cord (ASC) scaffold was prepared through chemical extraction, and in vivo promote spinal cord regeneration on lateral hemisected thoracic spinal cord in adult SD Rats. And to test the effect of ASC scaffold on functional recovery after spinal cord injury in vivo study by supporting adhesion, proliferation of host neural cells and axonal remyelination.
Methods:Twenty-four adult Sprague Dawley (SD) rats were conducted lateral hemisection in thoracic spinal cord to establish SCI model. The ASC scaffolds were implanted into the SCI lesion gaps in ASC group (n=6) while the lesion only as injury only group(n=6). The Basso, Beattie, and Bresnahan (BBB) locomotor test were conducted to assess neurologic function weekly for8weeks.14day and56day after SCI, six rats in each group were sacrificed and measured under histological, immunohistochemical and MBP examination.
Results:The ASC scaffold general looking is milky white, the lack of toughness and softness; The cross-section of it is a network structure of the extracellular matrix constituent, The most of the cells and myeloid have removed. Moreover, The ASC scaffold arranged in parallel fibrous structure and cross-section has a three dimensional network structure. In animal test the ASC scaffold could integrate well with the host spinal cord at the spinal cord gap and have beneficial effect on functional recovery (p<0.05). HE staining showed the group of spinal cord injury destroy the damaged spinal cord dorsal and ventral right part. The ASC scaffold graft group:The ASC scaffold have very good integration into the rat spinal cord hemisection defect and seen a large number of cell nuclei distributed in the ASC scaffold. Immunohistochemical showed few astrocytes was observed on ASC graft, forming an astrocyte-free area and, also, a few neural stem cells and neurons were observed on grafts. Among them, oligodendrocytes is main cell type, distributed in ASC graft at day56post-SCI. Some remyelination of ingrown axons had taken place inside in ASC scaffold.
Conclusion:ASC Scaffold, as a potential scaffold for SCI, could provide a microenvironment favorable to differentiation or proliferation of host oligodendrocyte and promote axon remyelination.
Chapter II Acellular Spinal Cord Scaffold Seeded with Mesenchymal Stem Cells Promotes Long-Distance Axon Regeneration and Functional Recovery in Spinal Cord Injured Rats
Objective:Stem cells based experimental therapies are partially successful for the recovery of Spinal Cord Injury (SCI). Recently, acellular spinal cord (ASC) scaffolds which mimic native extracellular matrix (ECM) have been successfully prepared. This study aimed to investigate whether the spinal cord lesion gap could be bridged by implantation of bionics designed ASC scaffold, either alone or seeded with human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), and their effects on functional improvement.
Methods:A laterally hemisected SCI lesion was performed in adult Sprague-Dawley (SD) rats (n=36) and ASC scaffolds were implanted into the lesion immediately, either with or without seeded hUCB-MSCs. All rats were behaviorally tested using the Basso-Beattie-Bresnahan (BBB) test once a week for8weeks. we detect nerve cells by immunofluorescence at second and eighth week after SCI, and completed BDA tracer at eighth week
Results:The three passgers of umbilical cord blood mesenchymal stem cells identified by flow cytometry:the cells with low expression of CD14(0.31%), CD34(0.82%), CD45(0.23%), HLA-DR (0.26%), and high expression following adhesion molecule CD90(99.99%), CD29(92.95%), CD44(99.98%) CD105(99.92%) and CD73(99.88%), and it is consistent with identification of mesenchymal stem cell phenotype reported before; human umbilical cord blood mesenchymal stem cells seeded in ASC scaffold were cultured in vitro for two days, and gross looking is white, soft and distribution of5-bromodeoxyuridine (Brdu)-positive human umbilical cord blood-derived mesenchymal stem cells in the scaffold of extracellular matrix composed of a three-dimensional network. Behavioral analysis showed that there was significant locomotor recovery improvement in combined treatment group (ASC scaffold and hUCB-MSCs) as compared with the control SCI only group (no scaffold)(p<0.01). Brdu-labeled hUCB-MSCs could also be observed in the implanted ASC two weeks after implantation. Moreover, host neural cells (mainly oligodendrocytes) were able to migrate into the graft. Biotin-dextran-amine (BDA) tracing test demonstrated that myelinated axons successfully grew into the graft and subsequently promoted axonal regeneration at lesion sites.
Conclusion:Our study provides the first experimental evidence that ASC scaffold seeded with hUCB-MSCs is able to bridge a spinal cord cavity and promote long-distance axon regeneration and functional recovery in SCI rats
Chapter Ⅲ The local immune regulation and apoptosis research on Acellular Spinal Cord Scaffold Seeded with Mesenchymal Stem Cells to repair spinal cord hemisection defects
Objective:explore the apoptosis and immune regulation mechanisms of rat spinal cord hemisection model of spinal cord injury repaired by Acellular Spinal Cord Scaffold Seeded with Mesenchymal Stem Cells
Method:Spinal cord acellular scaffold seed with human umbilical cord blood-derived mesenchymal stem cells as graft.36adult SD rats were randomized to three groups:group A, the SCI only group (n=12); group B, SCI+ASC scaffold (n=12); group C, SCI+ASC+hUCB-MSCs (n=12); The rat underwent spinal cord hemisection respectively implanted corresponding group. Two weeks after operation, Three animals of each group detect markers of immune cells and Tunel-positive cells by immunofluorescence and count positive cells; another three animals in each group under caspase-3activation degree detection, counting the OD value. Using analysis of variance to test the statistical differences in each group the number of positive cells and caspase-3activation OD values, differences between groups of homogeneity of variance with LSD test, P<0.05indicates statistical difference.
Results:two weeks after operation the group of SCI+ASC+hUCB-MSCs significantly inhibited Tunel positive cells (P<0.05) and caspase-3activation (P <0.05), it means this group inhibit apoptosis of local neural cells; On the contrary, the ASC scaffold group did not inhibit apoptosis. Moreover, the group of SCI+ASC+hUCB-MSCs significantly inhibited neutrophil (P<0.05), macrophages (microglia)(P<0.05), T lymphocytes (P<0.05) proliferation in spinal cord injury area. While, The ASC scaffold group doesn't significant inhibit immune response, It compared with SCI only group that there is no obvious differences on inflammatory positive cells (P>0.05). Furthermore the three groups was no significant difference on1gM positive expression (P>0.05).
Conclusion:The Acellular Spinal Cord Scaffold Seeded with Mesenchymal Stem Cells to repair spinal cord hemisection defects promote founction recovery through suppression of local immune cells, and inhibit apoptosis.
引文
[1]ROSSI S L, KEIRSTEAD H S. Stem cells and spinal cord regeneration [J]. Current opinion in biotechnology,2009,20(5):552-62.
[2]DOBKIN B H. Recommendations for publishing case studies of cell transplantation for spinal cord injury [J]. Neurorehabilitation and neural repair,2010, 24(8):687-91.
[3]NEWCOMB J D, WILLING A E, SANBERG P R. Umbilical cord blood cells [J]. Methods Mol Biol,2009,549(119-36.
[4]KUH S U, CHO Y E, YOON D H, et al. Functional recovery after human umbilical cord blood cells transplantation with brain-derived neutrophic factor into the spinal cord injured rat [J]. Acta neurochirurgica,2005,147(9):985-92; discussion 92.
[5]LEE J H, CHANG H S, KANG E H, et al. Percutaneous transplantation of human umbilical cord blood-derived multipotent stem cells in a canine model of spinal cord injury [J]. Journal of neurosurgery Spine,2009,11(6):749-57.
[6]VEERAVALLI K K, DASARI V R, TSUNG A J, et al. Human umbilical cord blood stem cells upregulate matrix metalloproteinase-2 in rats after spinal cord injury [J]. Neurobiology of disease,2009,36(1):200-12.
[7]CHUA S J, BIELECKI R, YAMANAKA N, et al. The effect of umbilical cord blood cells on outcomes after experimental traumatic spinal cord injury [J]. Spine, 2010,35(16):1520-6.
[8]DASARI V R, SPOMAR D G, LI L, et al. Umbilical cord blood stem cell mediated downregulation of fas improves functional recovery of rats after spinal cord injury [J]. Neurochemical research,2008,33(1):134-49.
[9]YAN-WU G, YI-QUAN K, MING L, et al. Human umbilical cord-derived Schwann-like cell transplantation combined with neurotrophin-3 administration in dyskinesia of rats with spinal cord injury [J]. Neurochemical research,2011,36(5): 783-92.
[10]CHO S R, YANG M S, YIM S H, et al. Neurally induced umbilical cord blood cells modestly repair injured spinal cords [J]. NeuroReport,2008,19(13):1259-63.
[11]LOW C B, LIOU Y C, TANG B L. Neural differentiation and potential use of stem cells from the human umbilical cord for central nervous system transplantation therapy [J]. Journal of neuroscience research,2008,86(8):1670-9.
[12]JEONG J A, GANG E J, HONG S H, et al. Rapid neural differentiation of human cord blood-derived mesenchymal stem cells [J]. NeuroReport,2004,15(11):1731-4.
[13]ARIEN-ZAKAY H, LAZAROVICI P, NAGLER A. Tissue regeneration potential in human umbilical cord blood [J]. Best practice & research Clinical haematology, 2010,23(2):291-303.
[14]ZHANG X Y, XUE H, LIU J M, et al. Chemically extracted acellular muscle:a new potential scaffold for spinal cord injury repair [J]. Journal of biomedical materials research Part A,2012,100(3):578-87.
[15]ITOSAKAH, KURODA S, SHICHINOHE H, et al. Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering [J]. Neuropathology:official journal of the Japanese Society of Neuropathology,2009,29(3):248-57.
[16]JOHNSON P J, PARKER S R, SAKIYAMA-ELBERT S E. Fibrin-based tissue engineering scaffolds enhance neural fiber sprouting and delay the accumulation of reactive astrocytes at the lesion in a subacute model of spinal cord injury [J]. Journal of biomedical materials research Part A,2010,92(1):152-63.
[17]HEJCL A, SEDY J, KAPCALOVA M, et al. HPMA-RGD hydrogels seeded with mesenchymal stem cells improve functional outcome in chronic spinal cord injury [J]. Stem cells and development,2010,19(10):1535-46.
[18]PRITCHARD C D, SLOTKIN J R, YU D, et al. Establishing a model spinal cord injury in the African green monkey for the preclinical evaluation of biodegradable polymer scaffolds seeded with human neural stem cells [J]. Journal of neuroscience methods,2010,188(2):258-69.
[19]GUO S Z, REN X J, WU B, et al. Preparation of the acellular scaffold of the spinal cord and the study of biocompatibility [J]. Spinal cord,2010,48(7):576-81.
[1]ROSSI S L, KEIRSTEAD H S. Stem cells and spinal cord regeneration [J]. Current opinion in biotechnology,2009,20(5):552-62.
[2]ENZMANN G U, BENTON R L, TALBOTT J F, et al. Functional considerations of stem cell transplantation therapy for spinal cord repair [J]. Journal of neurotrauma, 2006,23(3-4):479-95.
[3]BAN D X, KONG X H, FENG S Q, et al. Intraspinal cord graft of autologous activated Schwann cells efficiently promotes axonal regeneration and functional recovery after rat's spinal cord injury [J]. Brain research,2009,1256(149-61.
[4]HEJCL A, SEDY J, KAPCALOVA M, et al. HPMA-RGD hydrogels seeded with mesenchymal stem cells improve functional outcome in chronic spinal cord injury [J]. Stem cells and development,2010,19(10):1535-46.
[5]ITOSAKA H, KURODA S, SHICHINOHE H, et al. Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering [J]. Neuropathology:official journal of the Japanese Society of Neuropathology,2009,29(3):248-57.
[6]ZHANG X Y, XUE H, LIU J M, et al. Chemically extracted acellular muscle:a new potential scaffold for spinal cord injury repair [J]. Journal of biomedical materials research Part A,2012,100(3):578-87.
[7]GUO S Z, REN X J, WU B, et al. Preparation of the acellular scaffold of the spinal cord and the study of biocompatibility [J]. Spinal cord,2010,48(7):576-81.
[8]DINH P, BHATIAN, RASOULI A, et al. Transplantation of preconditioned Schwann cells following hemisection spinal cord injury [J]. Spine,2007,32(9): 943-9.
[9]BASSO D M, BEATTIE M S, BRESNAHAN J C. A sensitive and reliable locomotor rating scale for open field testing in rats [J]. Journal of neurotrauma,1995, 12(1):1-21.
[10]HURTADO A, MOON L D, MAQUET V, et al. Poly (D,L-lactic acid) macroporous guidance scaffolds seeded with Schwann cells genetically modified to secrete a bi-functional neurotrophin implanted in the completely transected adult rat thoracic spinal cord [J]. Biomaterials,2006,27(3):430-42.
[11]COMOLLI N, NEUHUBER B, FISCHER I, et al. In vitro analysis of PNIPAAm-PEG, a novel, injectable scaffold for spinal cord repair [J]. Acta biomaterialia,2009,5(4):1046-55.
[12]JOHNSON P J, PARKER S R, SAKIYAMA-ELBERT S E. Fibrin-based tissue engineering scaffolds enhance neural fiber sprouting and delay the accumulation of reactive astrocytes at the lesion in a subacute model of spinal cord injury [J]. Journal of biomedical materials research Part A,2010,92(1):152-63.
[13]EAST E, GOLDING J P, PHILLIPS J B. Engineering an Integrated Cellular Interface in Three-Dimensional Hydrogel Cultures Permits Monitoring of Reciprocal Astrocyte andNeuronal Responses (*) [J]. Tissue engineering Part C, Methods,2012,
[14]MCDONALD J W, BELEGU V. Demyelination and remyelination after spinal cord injury [J]. Journal of neurotrauma,2006,23(3-4):345-59.
[15]KOSHIZUKA S, OKADA S, OKAWA A, et al. Transplanted hematopoietic stem cells from bone marrow differentiate into neural lineage cells and promote functional recovery after spinal cord injury in mice [J]. Journal of neuropathology and experimental neurology,2004,63(1):64-72.
[16]CAO Q L, HOWARD R M, DENNISON J B, et al. Differentiation of engrafted neuronal-restricted precursor cells is inhibited in the traumatically injured spinal cord [J]. Experimental neurology,2002,177(2):349-59.
[17]ANDRESSEN C, ADRIAN S, FASSLER R, et al. The contribution of betal integrins to neuronal migration and differentiation depends on extracellular matrix molecules [J]. European journal of cell biology,2005,84(12):973-82.
[18]SZYMCZAK P, WOJCIK-STANASZEK L, SYPECKA J, et al. Effect of matrix metalloproteinases inhibition on the proliferation and differentiation of HUCB-NSCs cultured in the presence of adhesive substrates [J]. Acta neurobiologiae experimentalis,2010,70(4):325-36.
[19]MURASE S, HORWITZ A F. Deleted in colorectal carcinoma and differentially expressed integrins mediate the directional migration of neural precursors in the rostral migratory stream [J]. The Journal of neuroscience:the official journal of the Society for Neuroscience,2002,22(9):3568-79.
[20]MANNELLO F, TONTI G A, BAGNARA G P, et al. Role and function of matrix metalloproteinases in the differentiation and biological characterization of mesenchymal stem cells [J]. Stem Cells,2006,24(3):475-81.
[1]ROSSI S L, KEIRSTEAD H S. Stem cells and spinal cord regeneration [J]. Current opinion in biotechnology,2009,20(5):552-62.
[2]ENZMANN G U, BENTON R L, TALBOTT J F, et al. Functional considerations of stem cell transplantation therapy for spinal cord repair [J]. Journal of neurotrauma, 2006,23(3-4):479-95.
[3]BAN D X, KONG X H, FENG S Q, et al. Intraspinal cord graft of autologous activated Schwann cells efficiently promotes axonal regeneration and functional recovery after rat's spinal cord injury [J]. Brain research,2009,1256(149-61.
[4]CHO S R, YANG M S, YIM S H, et al. Neurally induced umbilical cord blood cells modestly repair injured spinal cords [J]. NeuroReport,2008,19(13):1259-63.
[5]DOBKIN B H. Recommendations for publishing case studies of cell transplantation for spinal cord injury [J]. Neurorehabilitation and neural repair,2010, 24(8):687-91.
[6]KUH S U, CHO Y E, YOON D H, et al. Functional recovery after human umbilical cord blood cells transplantation with brain-derived neutrophic factor into the spinal cord injured rat [J]. Acta neurochirurgica,2005,147(9):985-92; discussion 92.
[7]ARIEN-ZAKAY H, LAZAROVICI P, NAGLER A. Tissue regeneration potential in human umbilical cord blood [J]. Best practice & research Clinical haematology, 2010,23(2):291-303.
[8]MALGIERI A, KANTZARI E, PATRIZI M P, et al. Bone marrow and umbilical cord blood human mesenchymal stem cells:state of the art [J]. International journal of clinical and experimental medicine,2010,3(4):248-69.
[9]NEUHOFF S, MOERS J, RIEKS M, et al. Proliferation, differentiation, and cytokine secretion of human umbilical cord blood-derived mononuclear cells in vitro [J]. Experimental hematology,2007,35(7):1119-31.
[10]HILFIKER A, KASPER C, HASS R, et al. Mesenchymal stem cells and progenitor cells in connective tissue engineering and regenerative medicine:is there a future for transplantation? [J]. Langenbeck's archives of surgery/Deutsche Gesellschaft fur Chirurgie,2011,396(4):489-97.
[11]KERN S, EICHLER H, STOEVE J, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue [J]. Stem Cells, 2006,24(5):1294-301.
[12]LEE O K, KUO T K, CHEN W M, et al. Isolation of multipotent mesenchymal stem cells from umbilical cord blood [J]. Blood,2004,103(5):1669-75.
[13]WAGNER W, WEIN F, SECKINGER A, et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood [J]. Experimental hematology,2005,33(11):1402-16.
[14]MONTESINOS J J, FLORES-FIGUEROA E, CASTILLO-MEDINA S, et al. Human mesenchymal stromal cells from adult and neonatal sources:comparative analysis of their morphology, immunophenotype, differentiation patterns and neural protein expression [J]. Cytotherapy,2009,11(2):163-76.
[15]SUN W, BUZANSKA L, DOMANSKA-JANIK K, et al. Voltage-sensitive and ligand-gated channels in differentiating neural stem-like cells derived from the nonhematopoietic fraction of human umbilical cord blood [J]. Stem Cells,2005,23(7): 931-45.
[16]JIN W, XING Y Q, YANG A H. Epidermal growth factor promotes the differentiation of stem cells derived from human umbilical cord blood into neuron-like cells via taurine induction in vitro [J]. In vitro cellular & developmental biology Animal,2009,45(7):321-7.
[17]LIM J Y, PARK S I, OH J H, et al. Brain-derived neurotrophic factor stimulates the neural differentiation of human umbilical cord blood-derived mesenchymal stem cells and survival of differentiated cells through MAPK/ERK and PI3K/Akt-dependent signaling pathways [J]. Journal of neuroscience research,2008, 86(10):2168-78.
[18]JEONG J A, GANG E J, HONG S H, et al. Rapid neural differentiation of human cord blood-derived mesenchymal stem cells [J].NeuroReport,2004,15(11):1731-4.
[19]LEE M W, MOON Y J, YANG M S, et al. Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood [J]. Biochemical and biophysical research communications,2007,358(2):637-43.
[20]CAN A, KARAHUSEYINOGLU S. Concise review:human umbilical cord stroma with regard to the source of fetus-derived stem cells [J]. Stem Cells,2007, 25(11):2886-95.
[21]WEISS M L, TROYER D L. Stem cells in the umbilical cord [J]. Stem cell reviews,2006,2(2):155-62.
[22]LU L L, LIU Y J, YANG S G, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials [J]. Haematologica,2006,91(8):1017-26.
[23]MOEZZI L, POURFATHOLLAH A A, ALIMOGHADDAM K, et al. The effect of cryopreservation on clonogenic capacity and in vitro expansion potential of umbilical cord blood progenitor cells [J]. Transplantation proceedings,2005,37(10): 4500-3.
[24]GLENNIE S, SOEIRO I, DYSON P J, et al. Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells [J]. Blood,2005,105(7): 2821-7.
[25]KODE J A, MUKHERJEE S, JOGLEKAR M V, et al. Mesenchymal stem cells: immunobiology and role in immunomodulation and tissue regeneration [J]. Cytotherapy,2009,11(4):377-91.
[26]ABUMAREE M, AL JUMAH M, PACE R A, et al. Immunosuppressive Properties of Mesenchymal Stem Cells [J]. Stem cell reviews,2011,
[27]LOW C B, LIOU Y C, TANG B L. Neural differentiation and potential use of stem cells from the human umbilical cord for central nervous system transplantation therapy [J]. Journal of neuroscience research,2008,86(8):1670-9.
[28]CHUA S J, BIELECKI R, YAMANAKA N, et al. The effect of umbilical cord blood cells on outcomes after experimental traumatic spinal cord injury [J]. Spine, 2010,35(16):1520-6.
[29]DASARI V R, SPOMAR D G, LI L, et al. Umbilical cord blood stem cell mediated downregulation of fas improves functional recovery of rats after spinal cord injury [J].Neurochemical research,2008,33(1):134-49.
[30]VEERAVALLI K K, DASARI V R, TSUNG A J, et al. Human umbilical cord blood stem cells upregulate matrix metalloproteinase-2 in rats after spinal cord injury [J]. Neurobiology of disease,2009,36(1):200-12.
[31]ZHANG X Y, XUE H, LIU J M, et al. Chemically extracted acellular muscle:a new potential scaffold for spinal cord injury repair [J]. Journal of biomedical materials research Part A,2012,100(3):578-87.
[32]HEJCL A, SEDY J, KAPCALOVA M, et al. HPMA-RGD hydrogels seeded with mesenchymal stem cells improve functional outcome in chronic spinal cord injury [J]. Stem cells and development,2010,19(10):1535-46.
[33]PRITCHARD C D, SLOTKIN J R, YU D, et al. Establishing a model spinal cord injury in the African green monkey for the preclinical evaluation of biodegradable polymer scaffolds seeded with human neural stem cells [J]. Journal of neuroscience methods,2010,188(2):258-69.
[34]GUO S Z, REN X J, WU B, et al. Preparation of the acellular scaffold of the spinal cord and the study of biocompatibility [J]. Spinal cord,2010,48(7):576-81.
[35]NEWCOMB J D, WILLING A E, SANBERG P R. Umbilical cord blood cells [J]. Methods Mol Biol,2009,549(119-36.
[36]SEO Y, YANG S R, JEE M K, et al. Human umbilical cord blood-derived mesenchymal stem cells protect against neuronal cell death and ameliorate motor deficits in Niemann Pick type C1 mice [J]. Cell transplantation,2011,20(7):1033-47.
[37]GANG E J, JEONG J A, HAN S, et al. In vitro endothelial potential of human UC blood-derived mesenchymal stem cells [J]. Cytotherapy,2006,8(3):215-27.
[38]ITOSAKA H, KURODA S, SHICHINOHE H, et al. Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering [J]. Neuropathology:official journal of the Japanese Society of Neuropathology,2009,29(3):248-57.
[39]BASSO D M, BEATTIE M S, BRESNAHAN J C. A sensitive and reliable locomotor rating scale for open field testing in rats [J]. Journal of neurotrauma,1995, 12(1):1-21.
[40]ROMANOV Y A, SVINTSITSKAYA V A, SMIRNOV V N. Searching for alternative sources of postnatal human mesenchymal stem cells:candidate MSC-like cells from umbilical cord [J]. Stem Cells,2003,21(1):105-10.
[41]WEISS M L, MITCHELL K E, HIX J E, et al. Transplantation of porcine umbilical cord matrix cells into the rat brain [J]. Experimental neurology,2003, 182(2):288-99.
[42]WANG H S, HUNG S C, PENG S T, et al. Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord [J]. Stem Cells,2004,22(7):1330-7.
[43]SAPORTA S, KIM J J, WILLING A E, et al. Human umbilical cord blood stem cells infusion in spinal cord injury:engraftment and beneficial influence on behavior [J]. Journal of hematotherapy & stem cell research,2003,12(3):271-8.
[44]LIU C H, HWANG S M. Cytokine interactions in mesenchymal stem cells from cord blood [J]. Cytokine,2005,32(6):270-9.
[45]NEWMAN M B, WILLING A E, MANRESA J J, et al. Cytokines produced by cultured human umbilical cord blood (HUCB) cells:implications for brain repair [J]. Experimental neurology,2006,199(1):201-8.
[1]LI G L, FAROOQUE M, HOLTZ A, et al. Apoptosis of oligodendrocytes occurs for long distances away from the primary injury after compression trauma to rat spinal cord [J]. Acta neuropathologica,1999,98(5):473-80.
[2]KALOUS A, OSBORNE P B, KEAST J R. Acute and chronic changes in dorsal horn innervation by primary afferents and descending supraspinal pathways after spinal cord injury [J]. The Journal of comparative neurology,2007,504(3):238-53.
[3]QU W S, TIAN D S, GUO Z B, et al. Inhibition of EGFR/MAPK signaling reduces microglial inflammatory response and the associated secondary damage in rats after spinal cord injury [J]. Journal of neuroinflammation,2012,9(178.
[4]FONG B M, HAUPTMAN J S. From the bench to bedside:Secondary spinal cord injury, ischemic penumbra after stroke, neural regulation of appetite, microglia in Rett syndrome, signaling pathways in peripheral nerve regeneration [J]. Surgical neurology international,2012,3(56.
[5]OYINBO C A. Secondary injury mechanisms in traumatic spinal cord injury:a nugget of this multiply cascade [J]. Acta neurobiologiae experimentalis,2011,71(2): 281-99.
[6]ZIV Y, AVIDAN H, PLUCHINO S, et al. Synergy between immune cells and adult neural stem/progenitor cells promotes functional recovery from spinal cord injury [J]. Proceedings of the National Academy of Sciences of the United States of America,2006,103(35):13174-9.
[7]OLSON J K. Immune response by microglia in the spinal cord [J]. Annals of the New York Academy of Sciences,2010,1198(271-8.
[8]WEISHAUPT N, SILASI G, COLBOURNE F, et al. Secondary damage in the spinal cord after motor cortex injury in rats [J]. Journal of neurotrauma,2010,27(8): 1387-97.
[9]GAL P, KRAVCUKOVA P, MOKRY M, et al. Chemokines as possible targets in modulation of the secondary damage after acute spinal cord injury:a review [J]. Cellular and molecular neurobiology,2009,29(6-7):1025-35.
[10]HAINS B C, BLACK J A, WAXMAN S G. Primary cortical motor neurons undergo apoptosis after axotomizing spinal cord injury [J]. The Journal of comparative neurology,2003,462(3):328-41.
[11]SHIBUYA S, MIYAMOTO O, JANJUA N A, et al. Post-traumatic moderate systemic hypothermia reduces TUNEL positive cells following spinal cord injury in rat [J]. Spinal cord,2004,42(1):29-34.
[12]KAPTANOGLU E, CANER H, SOLAROGLU I, et al. Mexiletine treatment-induced inhibition of caspase-3 activation and improvement of behavioral recovery after spinal cord injury [J]. Journal of neurosurgery Spine,2005,3(1):53-6.
[13]SPRINGER J E, AZBILL R D, KNAPP P E. Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury [J]. Nature medicine,1999,5(8): 943-6.
[14]LIU L, PEI F X, TANG K L, et al. Expression and effect of Caspase-3 in neurons after tractive spinal cord injury in rats [J]. Chinese journal of traumatology= Zhonghua chuang shang za zhi/Chinese Medical Association,2005,8(4):220-4.
[15]ITOSAKA H, KURODA S, SHICHINOHE H, et al. Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering [J]. Neuropathology:official journal of the Japanese Society of Neuropathology,2009,29(3):248-57.
[16JDINH P, BHATIA N, RASOULI A, et al. Transplantation of preconditioned Schwann cells following hemisection spinal cord injury [J]. Spine,2007,32(9): 943-9.
[17]MUELLER C A, SCHLUESENER H J, CONRAD S, et al. Spinal cord injury-induced expression of the immune-regulatory chemokine interleukin-16 caused by activated microglia/macrophages and CD8+ cells [J]. Journal of neurosurgery Spine,2006,4(3):233-40.
[18]KURIHARA D, UENO M, TANAKA T, et al. Expression of galectin-1 in immune cells and glial cells after spinal cord injury [J]. Neuroscience research,2010, 66(3):265-70.
[19]CITRON B A, ARNOLD P M, HAYNES N Q et al. Neuroprotective effects of caspase-3 inhibition on functional recovery and tissue sparing after acute spinal cord injury [J]. Spine,2008,33(21):2269-77.
[20]KUBOTA K, SAIWAI H, KUMAMARU H, et al. Myeloperoxidase exacerbates secondary injury by generating highly reactive oxygen species and mediating neutrophil recruitment in experimental spinal cord injury [J]. Spine,2012,37(16): 1363-9.
[21]TJOA T, STRAUSBAUGH H J, MAIDA N, et al. The use of flow cytometry to assess neutrophil infiltration in the injured murine spinal cord [J]. Journal of neuroscience methods,2003,129(1):49-59.
[22]TAOKA Y, OKAJIMA K, MURAKAMI K, et al. Role of neutrophil elastase in compression-induced spinal cord injury in rats [J]. Brain research,1998,799(2): 264-9.
[23]SATO A, OHTAKI H, TSUMURAYA T, et al. Interleukin-1 participates in the classical and alternative activation of microglia/macrophages after spinal cord injury [J]. Journal of neuroinflammation,2012,9(65.
[24]WELLING L C, FIGUEIREDO E G. Microglia, corticotropin-releasing hormone, and spinal cord injury [J]. World neurosurgery,2010,74(4-5):388-9.
[25]BRADL M, BAUER J, FLUGEL A, et al. Complementary contribution of CD4 and CD8 T lymphocytes to T-cell infiltration of the intact and the degenerative spinal cord [J]. The American journal of pathology,2005,166(5):1441-50.
[26]WU B, MATIC D, DJOGO N, et al. Improved regeneration after spinal cord injury in mice lacking functional T-and B-lymphocytes [J]. Experimental neurology, 2012,237(2):274-85.
[27]GORFU G, VIRTANEN I, HUKKANEN M, et al. Laminin isoforms of lymph nodes and predominant role of alpha5-laminin(s) in adhesion and migration of blood lymphocytes [J]. Journal of leukocyte biology,2008,84(3):701-12.
[28]LINSLEY P S, CLARK E A, LEDBETTER J A. Pillars article:T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7/BB-1. 1990. Proc. Natl. Acad. Sci. USA 87:5031-5035 [J]. J Immunol,2009,182(5): 2559-63.
[29]OKADA S, SHIBA K, IWAMOTO Y. [Cell therapy for spinal cord injury] [J]. Fukuoka igaku zasshi= Hukuoka acta medica,2010,101(5):85-93.
[30]ZHOU C, YANG B, TIAN Y, et al. Immunomodulatory effect of human umbilical cord Wharton's jelly-derived mesenchymal stem cells on lymphocytes [J]. Cellular immunology,2011,272(1):33-8.
[31]WU L Q, BAI H, WANG C B, et al. [Effects of mesenchymal stem cells on proliferation and apoptosis of allogeneic B lymphocytes] [J]. Zhonghua yi xue za zhi, 2008,88(36):2562-5.
[32]YE F, QIAO Z H, ZHU L, et al. [Immunoregulatory effect of mesenchymal stem cells on active T lymphocytes] [J]. Zhongguo shi yan xue ye xue za zhi/Zhongguo bing li sheng li xue hui= Journal of experimental hematology/Chinese Association of Pathophysiology,2008,16(5):1116-20.
[1]ROMANOV Y A, SVINTSITSKAYA V A, SMIRNOV V N. Searching for alternative sources of postnatal human mesenchymal stem cells:candidate MSC-like cells from umbilical cord [J]. Stem Cells,2003,21(1):105-10.
[2]LU L L, LIU Y J, YANG S G, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials [J]. Haematologica,2006,91(8):1017-26.
[3]WEISS M L, MEDICETTY S, BLEDSOE A R, et al. Human umbilical cord matrix stem cells:preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease [J]. Stem Cells,2006,24(3):781-92.
[4]DESCHASEAUX F, SENSEBE L, HEYMANN D. Mechanisms of bone repair and regeneration [J]. Trends in molecular medicine,2009,15(9):417-29.
[5]JAGER M, JELINEK E M, WESS K M, et al. Bone marrow concentrate:a novel strategy for bone defect treatment [J]. Current stem cell research & therapy, 2009,4(1):34-43.
[6]LEE O K, KUO T K, CHEN W M, et al. Isolation of multipotent mesenchymal stem cells from umbilical cord blood [J]. Blood,2004,103(5):1669-75.
[7]KOGLER G, SENSKEN S, WERNET P. Comparative generation and characterization of pluripotent unrestricted somatic stem cells with mesenchymal stem cells from human cord blood [J]. Experimental hematology,2006,34(11): 1589-95.
[8]KOC O N, DAY J, NIEDER M, et al. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH) [J]. Bone marrow transplantation,2002,30(4):215-22.
[9]TONDREAU T, MEULEMAN N, DELFORGE A, et al. Mesenchymal stem cells derived from CD133-positive cells in mobilized peripheral blood and cord blood:proliferation, Oct4 expression, and plasticity [J]. Stem Cells,2005,23(8): 1105-12.
[10]FUCHS J R, HANNOUCHE D, TERADA S, et al. Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells [J]. Stem Cells,2005, 23(7):958-64.
[2]DOBKIN B H. Recommendations for publishing case studies of cell transplantation for spinal cord injury [J]. Neurorehabilitation and neural repair,2010, 24(8):687-91.
[3]NEWCOMB J D, WILLING A E, SANBERG P R. Umbilical cord blood cells [J]. Methods Mol Biol,2009,549(119-36.
[4]KUH S U, CHO Y E, YOON D H, et al. Functional recovery after human umbilical cord blood cells transplantation with brain-derived neutrophic factor into the spinal cord injured rat [J]. Acta neurochirurgica,2005,147(9):985-92; discussion 92.
[5]LEE J H, CHANG H S, KANG E H, et al. Percutaneous transplantation of human umbilical cord blood-derived multipotent stem cells in a canine model of spinal cord injury [J]. Journal of neurosurgery Spine,2009,11(6):749-57.
[6]VEERAVALLI K K, DASARI V R, TSUNG A J, et al. Human umbilical cord blood stem cells upregulate matrix metalloproteinase-2 in rats after spinal cord injury [J]. Neurobiology of disease,2009,36(1):200-12.
[7]CHUA S J, BIELECKI R, YAMANAKA N, et al. The effect of umbilical cord blood cells on outcomes after experimental traumatic spinal cord injury [J]. Spine, 2010,35(16):1520-6.
[8]DASARI V R, SPOMAR D G, LI L, et al. Umbilical cord blood stem cell mediated downregulation of fas improves functional recovery of rats after spinal cord injury [J]. Neurochemical research,2008,33(1):134-49.
[9]YAN-WU G, YI-QUAN K, MING L, et al. Human umbilical cord-derived Schwann-like cell transplantation combined with neurotrophin-3 administration in dyskinesia of rats with spinal cord injury [J]. Neurochemical research,2011,36(5): 783-92.
[10]CHO S R, YANG M S, YIM S H, et al. Neurally induced umbilical cord blood cells modestly repair injured spinal cords [J]. NeuroReport,2008,19(13):1259-63.
[11]LOW C B, LIOU Y C, TANG B L. Neural differentiation and potential use of stem cells from the human umbilical cord for central nervous system transplantation therapy [J]. Journal of neuroscience research,2008,86(8):1670-9.
[12]JEONG J A, GANG E J, HONG S H, et al. Rapid neural differentiation of human cord blood-derived mesenchymal stem cells [J]. NeuroReport,2004,15(11):1731-4.
[13]ARIEN-ZAKAY H, LAZAROVICI P, NAGLER A. Tissue regeneration potential in human umbilical cord blood [J]. Best practice & research Clinical haematology, 2010,23(2):291-303.
[14]ZHANG X Y, XUE H, LIU J M, et al. Chemically extracted acellular muscle:a new potential scaffold for spinal cord injury repair [J]. Journal of biomedical materials research Part A,2012,100(3):578-87.
[15]ITOSAKAH, KURODA S, SHICHINOHE H, et al. Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering [J]. Neuropathology:official journal of the Japanese Society of Neuropathology,2009,29(3):248-57.
[16]JOHNSON P J, PARKER S R, SAKIYAMA-ELBERT S E. Fibrin-based tissue engineering scaffolds enhance neural fiber sprouting and delay the accumulation of reactive astrocytes at the lesion in a subacute model of spinal cord injury [J]. Journal of biomedical materials research Part A,2010,92(1):152-63.
[17]HEJCL A, SEDY J, KAPCALOVA M, et al. HPMA-RGD hydrogels seeded with mesenchymal stem cells improve functional outcome in chronic spinal cord injury [J]. Stem cells and development,2010,19(10):1535-46.
[18]PRITCHARD C D, SLOTKIN J R, YU D, et al. Establishing a model spinal cord injury in the African green monkey for the preclinical evaluation of biodegradable polymer scaffolds seeded with human neural stem cells [J]. Journal of neuroscience methods,2010,188(2):258-69.
[19]GUO S Z, REN X J, WU B, et al. Preparation of the acellular scaffold of the spinal cord and the study of biocompatibility [J]. Spinal cord,2010,48(7):576-81.
[1]ROSSI S L, KEIRSTEAD H S. Stem cells and spinal cord regeneration [J]. Current opinion in biotechnology,2009,20(5):552-62.
[2]ENZMANN G U, BENTON R L, TALBOTT J F, et al. Functional considerations of stem cell transplantation therapy for spinal cord repair [J]. Journal of neurotrauma, 2006,23(3-4):479-95.
[3]BAN D X, KONG X H, FENG S Q, et al. Intraspinal cord graft of autologous activated Schwann cells efficiently promotes axonal regeneration and functional recovery after rat's spinal cord injury [J]. Brain research,2009,1256(149-61.
[4]HEJCL A, SEDY J, KAPCALOVA M, et al. HPMA-RGD hydrogels seeded with mesenchymal stem cells improve functional outcome in chronic spinal cord injury [J]. Stem cells and development,2010,19(10):1535-46.
[5]ITOSAKA H, KURODA S, SHICHINOHE H, et al. Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering [J]. Neuropathology:official journal of the Japanese Society of Neuropathology,2009,29(3):248-57.
[6]ZHANG X Y, XUE H, LIU J M, et al. Chemically extracted acellular muscle:a new potential scaffold for spinal cord injury repair [J]. Journal of biomedical materials research Part A,2012,100(3):578-87.
[7]GUO S Z, REN X J, WU B, et al. Preparation of the acellular scaffold of the spinal cord and the study of biocompatibility [J]. Spinal cord,2010,48(7):576-81.
[8]DINH P, BHATIAN, RASOULI A, et al. Transplantation of preconditioned Schwann cells following hemisection spinal cord injury [J]. Spine,2007,32(9): 943-9.
[9]BASSO D M, BEATTIE M S, BRESNAHAN J C. A sensitive and reliable locomotor rating scale for open field testing in rats [J]. Journal of neurotrauma,1995, 12(1):1-21.
[10]HURTADO A, MOON L D, MAQUET V, et al. Poly (D,L-lactic acid) macroporous guidance scaffolds seeded with Schwann cells genetically modified to secrete a bi-functional neurotrophin implanted in the completely transected adult rat thoracic spinal cord [J]. Biomaterials,2006,27(3):430-42.
[11]COMOLLI N, NEUHUBER B, FISCHER I, et al. In vitro analysis of PNIPAAm-PEG, a novel, injectable scaffold for spinal cord repair [J]. Acta biomaterialia,2009,5(4):1046-55.
[12]JOHNSON P J, PARKER S R, SAKIYAMA-ELBERT S E. Fibrin-based tissue engineering scaffolds enhance neural fiber sprouting and delay the accumulation of reactive astrocytes at the lesion in a subacute model of spinal cord injury [J]. Journal of biomedical materials research Part A,2010,92(1):152-63.
[13]EAST E, GOLDING J P, PHILLIPS J B. Engineering an Integrated Cellular Interface in Three-Dimensional Hydrogel Cultures Permits Monitoring of Reciprocal Astrocyte andNeuronal Responses (*) [J]. Tissue engineering Part C, Methods,2012,
[14]MCDONALD J W, BELEGU V. Demyelination and remyelination after spinal cord injury [J]. Journal of neurotrauma,2006,23(3-4):345-59.
[15]KOSHIZUKA S, OKADA S, OKAWA A, et al. Transplanted hematopoietic stem cells from bone marrow differentiate into neural lineage cells and promote functional recovery after spinal cord injury in mice [J]. Journal of neuropathology and experimental neurology,2004,63(1):64-72.
[16]CAO Q L, HOWARD R M, DENNISON J B, et al. Differentiation of engrafted neuronal-restricted precursor cells is inhibited in the traumatically injured spinal cord [J]. Experimental neurology,2002,177(2):349-59.
[17]ANDRESSEN C, ADRIAN S, FASSLER R, et al. The contribution of betal integrins to neuronal migration and differentiation depends on extracellular matrix molecules [J]. European journal of cell biology,2005,84(12):973-82.
[18]SZYMCZAK P, WOJCIK-STANASZEK L, SYPECKA J, et al. Effect of matrix metalloproteinases inhibition on the proliferation and differentiation of HUCB-NSCs cultured in the presence of adhesive substrates [J]. Acta neurobiologiae experimentalis,2010,70(4):325-36.
[19]MURASE S, HORWITZ A F. Deleted in colorectal carcinoma and differentially expressed integrins mediate the directional migration of neural precursors in the rostral migratory stream [J]. The Journal of neuroscience:the official journal of the Society for Neuroscience,2002,22(9):3568-79.
[20]MANNELLO F, TONTI G A, BAGNARA G P, et al. Role and function of matrix metalloproteinases in the differentiation and biological characterization of mesenchymal stem cells [J]. Stem Cells,2006,24(3):475-81.
[1]ROSSI S L, KEIRSTEAD H S. Stem cells and spinal cord regeneration [J]. Current opinion in biotechnology,2009,20(5):552-62.
[2]ENZMANN G U, BENTON R L, TALBOTT J F, et al. Functional considerations of stem cell transplantation therapy for spinal cord repair [J]. Journal of neurotrauma, 2006,23(3-4):479-95.
[3]BAN D X, KONG X H, FENG S Q, et al. Intraspinal cord graft of autologous activated Schwann cells efficiently promotes axonal regeneration and functional recovery after rat's spinal cord injury [J]. Brain research,2009,1256(149-61.
[4]CHO S R, YANG M S, YIM S H, et al. Neurally induced umbilical cord blood cells modestly repair injured spinal cords [J]. NeuroReport,2008,19(13):1259-63.
[5]DOBKIN B H. Recommendations for publishing case studies of cell transplantation for spinal cord injury [J]. Neurorehabilitation and neural repair,2010, 24(8):687-91.
[6]KUH S U, CHO Y E, YOON D H, et al. Functional recovery after human umbilical cord blood cells transplantation with brain-derived neutrophic factor into the spinal cord injured rat [J]. Acta neurochirurgica,2005,147(9):985-92; discussion 92.
[7]ARIEN-ZAKAY H, LAZAROVICI P, NAGLER A. Tissue regeneration potential in human umbilical cord blood [J]. Best practice & research Clinical haematology, 2010,23(2):291-303.
[8]MALGIERI A, KANTZARI E, PATRIZI M P, et al. Bone marrow and umbilical cord blood human mesenchymal stem cells:state of the art [J]. International journal of clinical and experimental medicine,2010,3(4):248-69.
[9]NEUHOFF S, MOERS J, RIEKS M, et al. Proliferation, differentiation, and cytokine secretion of human umbilical cord blood-derived mononuclear cells in vitro [J]. Experimental hematology,2007,35(7):1119-31.
[10]HILFIKER A, KASPER C, HASS R, et al. Mesenchymal stem cells and progenitor cells in connective tissue engineering and regenerative medicine:is there a future for transplantation? [J]. Langenbeck's archives of surgery/Deutsche Gesellschaft fur Chirurgie,2011,396(4):489-97.
[11]KERN S, EICHLER H, STOEVE J, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue [J]. Stem Cells, 2006,24(5):1294-301.
[12]LEE O K, KUO T K, CHEN W M, et al. Isolation of multipotent mesenchymal stem cells from umbilical cord blood [J]. Blood,2004,103(5):1669-75.
[13]WAGNER W, WEIN F, SECKINGER A, et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood [J]. Experimental hematology,2005,33(11):1402-16.
[14]MONTESINOS J J, FLORES-FIGUEROA E, CASTILLO-MEDINA S, et al. Human mesenchymal stromal cells from adult and neonatal sources:comparative analysis of their morphology, immunophenotype, differentiation patterns and neural protein expression [J]. Cytotherapy,2009,11(2):163-76.
[15]SUN W, BUZANSKA L, DOMANSKA-JANIK K, et al. Voltage-sensitive and ligand-gated channels in differentiating neural stem-like cells derived from the nonhematopoietic fraction of human umbilical cord blood [J]. Stem Cells,2005,23(7): 931-45.
[16]JIN W, XING Y Q, YANG A H. Epidermal growth factor promotes the differentiation of stem cells derived from human umbilical cord blood into neuron-like cells via taurine induction in vitro [J]. In vitro cellular & developmental biology Animal,2009,45(7):321-7.
[17]LIM J Y, PARK S I, OH J H, et al. Brain-derived neurotrophic factor stimulates the neural differentiation of human umbilical cord blood-derived mesenchymal stem cells and survival of differentiated cells through MAPK/ERK and PI3K/Akt-dependent signaling pathways [J]. Journal of neuroscience research,2008, 86(10):2168-78.
[18]JEONG J A, GANG E J, HONG S H, et al. Rapid neural differentiation of human cord blood-derived mesenchymal stem cells [J].NeuroReport,2004,15(11):1731-4.
[19]LEE M W, MOON Y J, YANG M S, et al. Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood [J]. Biochemical and biophysical research communications,2007,358(2):637-43.
[20]CAN A, KARAHUSEYINOGLU S. Concise review:human umbilical cord stroma with regard to the source of fetus-derived stem cells [J]. Stem Cells,2007, 25(11):2886-95.
[21]WEISS M L, TROYER D L. Stem cells in the umbilical cord [J]. Stem cell reviews,2006,2(2):155-62.
[22]LU L L, LIU Y J, YANG S G, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials [J]. Haematologica,2006,91(8):1017-26.
[23]MOEZZI L, POURFATHOLLAH A A, ALIMOGHADDAM K, et al. The effect of cryopreservation on clonogenic capacity and in vitro expansion potential of umbilical cord blood progenitor cells [J]. Transplantation proceedings,2005,37(10): 4500-3.
[24]GLENNIE S, SOEIRO I, DYSON P J, et al. Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells [J]. Blood,2005,105(7): 2821-7.
[25]KODE J A, MUKHERJEE S, JOGLEKAR M V, et al. Mesenchymal stem cells: immunobiology and role in immunomodulation and tissue regeneration [J]. Cytotherapy,2009,11(4):377-91.
[26]ABUMAREE M, AL JUMAH M, PACE R A, et al. Immunosuppressive Properties of Mesenchymal Stem Cells [J]. Stem cell reviews,2011,
[27]LOW C B, LIOU Y C, TANG B L. Neural differentiation and potential use of stem cells from the human umbilical cord for central nervous system transplantation therapy [J]. Journal of neuroscience research,2008,86(8):1670-9.
[28]CHUA S J, BIELECKI R, YAMANAKA N, et al. The effect of umbilical cord blood cells on outcomes after experimental traumatic spinal cord injury [J]. Spine, 2010,35(16):1520-6.
[29]DASARI V R, SPOMAR D G, LI L, et al. Umbilical cord blood stem cell mediated downregulation of fas improves functional recovery of rats after spinal cord injury [J].Neurochemical research,2008,33(1):134-49.
[30]VEERAVALLI K K, DASARI V R, TSUNG A J, et al. Human umbilical cord blood stem cells upregulate matrix metalloproteinase-2 in rats after spinal cord injury [J]. Neurobiology of disease,2009,36(1):200-12.
[31]ZHANG X Y, XUE H, LIU J M, et al. Chemically extracted acellular muscle:a new potential scaffold for spinal cord injury repair [J]. Journal of biomedical materials research Part A,2012,100(3):578-87.
[32]HEJCL A, SEDY J, KAPCALOVA M, et al. HPMA-RGD hydrogels seeded with mesenchymal stem cells improve functional outcome in chronic spinal cord injury [J]. Stem cells and development,2010,19(10):1535-46.
[33]PRITCHARD C D, SLOTKIN J R, YU D, et al. Establishing a model spinal cord injury in the African green monkey for the preclinical evaluation of biodegradable polymer scaffolds seeded with human neural stem cells [J]. Journal of neuroscience methods,2010,188(2):258-69.
[34]GUO S Z, REN X J, WU B, et al. Preparation of the acellular scaffold of the spinal cord and the study of biocompatibility [J]. Spinal cord,2010,48(7):576-81.
[35]NEWCOMB J D, WILLING A E, SANBERG P R. Umbilical cord blood cells [J]. Methods Mol Biol,2009,549(119-36.
[36]SEO Y, YANG S R, JEE M K, et al. Human umbilical cord blood-derived mesenchymal stem cells protect against neuronal cell death and ameliorate motor deficits in Niemann Pick type C1 mice [J]. Cell transplantation,2011,20(7):1033-47.
[37]GANG E J, JEONG J A, HAN S, et al. In vitro endothelial potential of human UC blood-derived mesenchymal stem cells [J]. Cytotherapy,2006,8(3):215-27.
[38]ITOSAKA H, KURODA S, SHICHINOHE H, et al. Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering [J]. Neuropathology:official journal of the Japanese Society of Neuropathology,2009,29(3):248-57.
[39]BASSO D M, BEATTIE M S, BRESNAHAN J C. A sensitive and reliable locomotor rating scale for open field testing in rats [J]. Journal of neurotrauma,1995, 12(1):1-21.
[40]ROMANOV Y A, SVINTSITSKAYA V A, SMIRNOV V N. Searching for alternative sources of postnatal human mesenchymal stem cells:candidate MSC-like cells from umbilical cord [J]. Stem Cells,2003,21(1):105-10.
[41]WEISS M L, MITCHELL K E, HIX J E, et al. Transplantation of porcine umbilical cord matrix cells into the rat brain [J]. Experimental neurology,2003, 182(2):288-99.
[42]WANG H S, HUNG S C, PENG S T, et al. Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord [J]. Stem Cells,2004,22(7):1330-7.
[43]SAPORTA S, KIM J J, WILLING A E, et al. Human umbilical cord blood stem cells infusion in spinal cord injury:engraftment and beneficial influence on behavior [J]. Journal of hematotherapy & stem cell research,2003,12(3):271-8.
[44]LIU C H, HWANG S M. Cytokine interactions in mesenchymal stem cells from cord blood [J]. Cytokine,2005,32(6):270-9.
[45]NEWMAN M B, WILLING A E, MANRESA J J, et al. Cytokines produced by cultured human umbilical cord blood (HUCB) cells:implications for brain repair [J]. Experimental neurology,2006,199(1):201-8.
[1]LI G L, FAROOQUE M, HOLTZ A, et al. Apoptosis of oligodendrocytes occurs for long distances away from the primary injury after compression trauma to rat spinal cord [J]. Acta neuropathologica,1999,98(5):473-80.
[2]KALOUS A, OSBORNE P B, KEAST J R. Acute and chronic changes in dorsal horn innervation by primary afferents and descending supraspinal pathways after spinal cord injury [J]. The Journal of comparative neurology,2007,504(3):238-53.
[3]QU W S, TIAN D S, GUO Z B, et al. Inhibition of EGFR/MAPK signaling reduces microglial inflammatory response and the associated secondary damage in rats after spinal cord injury [J]. Journal of neuroinflammation,2012,9(178.
[4]FONG B M, HAUPTMAN J S. From the bench to bedside:Secondary spinal cord injury, ischemic penumbra after stroke, neural regulation of appetite, microglia in Rett syndrome, signaling pathways in peripheral nerve regeneration [J]. Surgical neurology international,2012,3(56.
[5]OYINBO C A. Secondary injury mechanisms in traumatic spinal cord injury:a nugget of this multiply cascade [J]. Acta neurobiologiae experimentalis,2011,71(2): 281-99.
[6]ZIV Y, AVIDAN H, PLUCHINO S, et al. Synergy between immune cells and adult neural stem/progenitor cells promotes functional recovery from spinal cord injury [J]. Proceedings of the National Academy of Sciences of the United States of America,2006,103(35):13174-9.
[7]OLSON J K. Immune response by microglia in the spinal cord [J]. Annals of the New York Academy of Sciences,2010,1198(271-8.
[8]WEISHAUPT N, SILASI G, COLBOURNE F, et al. Secondary damage in the spinal cord after motor cortex injury in rats [J]. Journal of neurotrauma,2010,27(8): 1387-97.
[9]GAL P, KRAVCUKOVA P, MOKRY M, et al. Chemokines as possible targets in modulation of the secondary damage after acute spinal cord injury:a review [J]. Cellular and molecular neurobiology,2009,29(6-7):1025-35.
[10]HAINS B C, BLACK J A, WAXMAN S G. Primary cortical motor neurons undergo apoptosis after axotomizing spinal cord injury [J]. The Journal of comparative neurology,2003,462(3):328-41.
[11]SHIBUYA S, MIYAMOTO O, JANJUA N A, et al. Post-traumatic moderate systemic hypothermia reduces TUNEL positive cells following spinal cord injury in rat [J]. Spinal cord,2004,42(1):29-34.
[12]KAPTANOGLU E, CANER H, SOLAROGLU I, et al. Mexiletine treatment-induced inhibition of caspase-3 activation and improvement of behavioral recovery after spinal cord injury [J]. Journal of neurosurgery Spine,2005,3(1):53-6.
[13]SPRINGER J E, AZBILL R D, KNAPP P E. Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury [J]. Nature medicine,1999,5(8): 943-6.
[14]LIU L, PEI F X, TANG K L, et al. Expression and effect of Caspase-3 in neurons after tractive spinal cord injury in rats [J]. Chinese journal of traumatology= Zhonghua chuang shang za zhi/Chinese Medical Association,2005,8(4):220-4.
[15]ITOSAKA H, KURODA S, SHICHINOHE H, et al. Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering [J]. Neuropathology:official journal of the Japanese Society of Neuropathology,2009,29(3):248-57.
[16JDINH P, BHATIA N, RASOULI A, et al. Transplantation of preconditioned Schwann cells following hemisection spinal cord injury [J]. Spine,2007,32(9): 943-9.
[17]MUELLER C A, SCHLUESENER H J, CONRAD S, et al. Spinal cord injury-induced expression of the immune-regulatory chemokine interleukin-16 caused by activated microglia/macrophages and CD8+ cells [J]. Journal of neurosurgery Spine,2006,4(3):233-40.
[18]KURIHARA D, UENO M, TANAKA T, et al. Expression of galectin-1 in immune cells and glial cells after spinal cord injury [J]. Neuroscience research,2010, 66(3):265-70.
[19]CITRON B A, ARNOLD P M, HAYNES N Q et al. Neuroprotective effects of caspase-3 inhibition on functional recovery and tissue sparing after acute spinal cord injury [J]. Spine,2008,33(21):2269-77.
[20]KUBOTA K, SAIWAI H, KUMAMARU H, et al. Myeloperoxidase exacerbates secondary injury by generating highly reactive oxygen species and mediating neutrophil recruitment in experimental spinal cord injury [J]. Spine,2012,37(16): 1363-9.
[21]TJOA T, STRAUSBAUGH H J, MAIDA N, et al. The use of flow cytometry to assess neutrophil infiltration in the injured murine spinal cord [J]. Journal of neuroscience methods,2003,129(1):49-59.
[22]TAOKA Y, OKAJIMA K, MURAKAMI K, et al. Role of neutrophil elastase in compression-induced spinal cord injury in rats [J]. Brain research,1998,799(2): 264-9.
[23]SATO A, OHTAKI H, TSUMURAYA T, et al. Interleukin-1 participates in the classical and alternative activation of microglia/macrophages after spinal cord injury [J]. Journal of neuroinflammation,2012,9(65.
[24]WELLING L C, FIGUEIREDO E G. Microglia, corticotropin-releasing hormone, and spinal cord injury [J]. World neurosurgery,2010,74(4-5):388-9.
[25]BRADL M, BAUER J, FLUGEL A, et al. Complementary contribution of CD4 and CD8 T lymphocytes to T-cell infiltration of the intact and the degenerative spinal cord [J]. The American journal of pathology,2005,166(5):1441-50.
[26]WU B, MATIC D, DJOGO N, et al. Improved regeneration after spinal cord injury in mice lacking functional T-and B-lymphocytes [J]. Experimental neurology, 2012,237(2):274-85.
[27]GORFU G, VIRTANEN I, HUKKANEN M, et al. Laminin isoforms of lymph nodes and predominant role of alpha5-laminin(s) in adhesion and migration of blood lymphocytes [J]. Journal of leukocyte biology,2008,84(3):701-12.
[28]LINSLEY P S, CLARK E A, LEDBETTER J A. Pillars article:T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7/BB-1. 1990. Proc. Natl. Acad. Sci. USA 87:5031-5035 [J]. J Immunol,2009,182(5): 2559-63.
[29]OKADA S, SHIBA K, IWAMOTO Y. [Cell therapy for spinal cord injury] [J]. Fukuoka igaku zasshi= Hukuoka acta medica,2010,101(5):85-93.
[30]ZHOU C, YANG B, TIAN Y, et al. Immunomodulatory effect of human umbilical cord Wharton's jelly-derived mesenchymal stem cells on lymphocytes [J]. Cellular immunology,2011,272(1):33-8.
[31]WU L Q, BAI H, WANG C B, et al. [Effects of mesenchymal stem cells on proliferation and apoptosis of allogeneic B lymphocytes] [J]. Zhonghua yi xue za zhi, 2008,88(36):2562-5.
[32]YE F, QIAO Z H, ZHU L, et al. [Immunoregulatory effect of mesenchymal stem cells on active T lymphocytes] [J]. Zhongguo shi yan xue ye xue za zhi/Zhongguo bing li sheng li xue hui= Journal of experimental hematology/Chinese Association of Pathophysiology,2008,16(5):1116-20.
[1]ROMANOV Y A, SVINTSITSKAYA V A, SMIRNOV V N. Searching for alternative sources of postnatal human mesenchymal stem cells:candidate MSC-like cells from umbilical cord [J]. Stem Cells,2003,21(1):105-10.
[2]LU L L, LIU Y J, YANG S G, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials [J]. Haematologica,2006,91(8):1017-26.
[3]WEISS M L, MEDICETTY S, BLEDSOE A R, et al. Human umbilical cord matrix stem cells:preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease [J]. Stem Cells,2006,24(3):781-92.
[4]DESCHASEAUX F, SENSEBE L, HEYMANN D. Mechanisms of bone repair and regeneration [J]. Trends in molecular medicine,2009,15(9):417-29.
[5]JAGER M, JELINEK E M, WESS K M, et al. Bone marrow concentrate:a novel strategy for bone defect treatment [J]. Current stem cell research & therapy, 2009,4(1):34-43.
[6]LEE O K, KUO T K, CHEN W M, et al. Isolation of multipotent mesenchymal stem cells from umbilical cord blood [J]. Blood,2004,103(5):1669-75.
[7]KOGLER G, SENSKEN S, WERNET P. Comparative generation and characterization of pluripotent unrestricted somatic stem cells with mesenchymal stem cells from human cord blood [J]. Experimental hematology,2006,34(11): 1589-95.
[8]KOC O N, DAY J, NIEDER M, et al. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH) [J]. Bone marrow transplantation,2002,30(4):215-22.
[9]TONDREAU T, MEULEMAN N, DELFORGE A, et al. Mesenchymal stem cells derived from CD133-positive cells in mobilized peripheral blood and cord blood:proliferation, Oct4 expression, and plasticity [J]. Stem Cells,2005,23(8): 1105-12.
[10]FUCHS J R, HANNOUCHE D, TERADA S, et al. Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells [J]. Stem Cells,2005, 23(7):958-64.