脊髓损伤的细胞治疗策略

脊髓损伤（spinal cord injury,SCI）是一种以脊髓长轴索被破坏为主并伴有功能障碍的神经系统疾病,其修复困难,预后不理想,SCI治疗是临床上的一个挑战。SCI发生后,损伤处可见囊性的空腔,发生胶质瘢痕、髓鞘抑制物和炎症等一系列复杂的生理过程,对SCI的治疗非常不利,是目前治疗中难以克服的问题。通过神经再生重建通路以及通过神经保护抑制继发性损伤是当前SCI治疗的两个主要策略。神经再生的应用方法有组织工程和细胞移植。对于神经保护,目前主要是应用药物疗法。细胞治疗可通过细胞移植修复SCI,具有神经保护、免疫调节、轴突再生、神经元接力形成和再髓鞘化等优点。本文将综述不同类型的细胞治疗在SCI修复中的作用。     

脊髓损伤后髓鞘相关抑制因子的作用机制及基因治疗现状

脊髓损伤(SCI)是一种严重威胁人类健康的疾病,可致损伤平面以下的运动、感觉及植物神经功能障碍,致残率高,给社会和家庭带来沉重的负担.其治疗一直是困扰医学界的一大难题.和外周神经系统(PNS)相比,中枢神经系统(CNS)的脊髓损伤后不能再生的原因主要由于:(1)脊髓髓磷脂的几个糖蛋白成份对神经轴突再生是抑制的;(2)脊髓对损伤的生理反应与周围神经不同,脊髓损伤后,损伤部位的巨噬细胞浸润较慢,使抑制性的髓磷脂清除延迟,这主要是血-脊髓屏障的存在;(3)损伤脊髓的远端不能像周围神经一样在损伤后上调表达细胞粘附分子;(4)脊髓内星形胶质细胞扩增,变成反应性星形胶质细胞,产生抑制神经再生的胶质瘢痕.归结起来主要是两方面的作用:(1)脊髓轴突神经元本身缺乏再生的能力;(2)它们的再生被抑制性的脊髓微环境所抑制.后者在其中起了更为关键的作用,脊髓髓鞘含有丰富的轴突生长抑制因子,主要有:髓鞘相关糖蛋白( myelin-associated glycoprotein,MAG),少突胶质细胞-髓鞘糖蛋白(oligodendrocyte-myelin glycoprotein,OMgp),Nogo -A及ephrin - B3,统称为髓鞘相关抑制因子(myelin-associated inhibitor factors,MAIFs[1]),MAG,OMgp和Nogo-A虽然结构不同,但都与相同的MAIFs受体复合体- Nogo受体复合体(NgR1/p75/LIN - GO -1复合体)结合发挥作用,MAIFs的相继发现及对其作用机制逐步深入了解使围绕髓鞘抑制因子的研究成为中枢神经系统损伤后神经轴突再生研究的热点.     

髓磷脂相关衍生物与脊髓损伤修复研究进展

成年哺乳动物脊髓及周围神经损伤后轴突再生受限的主要因素之一是髓鞘产生的抑制神经生长相关因子.针对不同的神经元细胞及其所处的微环境,髓磷脂相关衍生物以不同区域位点与对应受体相互作用,产生的抑制作用及程度亦不同.应用髓磷脂相关衍生物抑制剂或其受体阻断剂可在一定程度上增加轴突再生能力.目前国内外公认的髓磷脂相关神经再生抑制物有勿动蛋白、髓磷脂相关糖蛋白、少突细胞髓鞘相关糖蛋白、硫酸软骨素蛋白聚糖及新近发现的ephrin-B3等物质.该文就髓磷脂相关衍生物与脊髓损伤后再生的相关研究作一综述,通过对上述各物质微观结构及分布特点的研究,探讨在不同微环境或与其他物质共存条件下,髓磷脂相关衍生物抑制脊髓损伤后再生的机制.     

脊髓损伤后神经元铁死亡的分子调控机制及应用研究进展

脊髓损伤（spinal cord injury,SCI）主要分为原发性和继发性损伤。SCI比较难治疗的原因很大程度上是其继发性损伤带来的一系列复杂的级联放大反应,涉及的机制也非常多,包括炎症反应、氧自由基损伤、谷氨酸中毒、钙超载、线粒体功能障碍、轴突脱髓鞘、坏死和凋亡等;由于SCI的原发性损伤往往是不可逆转的,所以现在的大多数研究都主要集中在逆转继发性损伤中的神经元死亡和变性,促进轴突和髓鞘的再生。而近年来研究发现SCI与铁死亡之间也有着密切联系,抑制SCI后的神经元铁死亡可以保护神经元。鉴于此,笔者对SCI后神经元铁死亡的分子调控机制及其研究进展进行综述,以期为调节神经元铁死亡治疗SCI提供参考。     

大鼠臂丛神经根性回植后脊髓病理改变和轴突再生

目的 探讨臂丛神经根性撕脱后神经根再植入脊髓的可行性。方法 采用大鼠颈5-7，神经根性撕脱伤实验动物模型，伤后将C5-7，神经根即刻植入脊髓。分别于神经根植入后3周、3个月、6个月取材。应用组织病理活检、免疫组化技术及神经示踪技术，对神经中枢及吻合口下段神经干检查。观察脊髓前角运动神经元和神经元内尼氏体数目和形态的改变；周围神经纤维再生数目、距离，轴索和髓鞘发育情况。结果 臂丛神经根性撕脱伤对动物生长和存活有较大的影响。脊髓前角运动神经元数目在3个月内持续减少，3个月后趋于稳定，6个月时脊髓前角大型运动细胞坏死比率在40％左右，残存的神经元多为受损的神经元，尼氏体减少或消失。脊髓前角运动神经元再生轴突可重新生长入周围神经干，再生神经纤维轴索较细，大部分髓鞘发育不完全，轴突再生距离较短，肌皮神经6个月内无神经纤维再生。结论 臂丛神经根性撕脱伤，神经根回植入脊髓后，脊髓前角运动神经元坏死比率为40％左右，残存神经元多为受损神经元，再生神经纤维表现为动力不足和发育不全，对终末器官功能恢复没有意义。     

血管内皮生长因子对神经系统血管再生与神经营养作用研究进展

近年来一系列研究表明,血管内皮生长因子除了具有促进新生血管形成的主要作用外,还可以直接作用于神经元及神经胶质细胞,刺激它们生长、存活,促进轴突再生。因为这种双重特性,它在一些神经系统疾病,如脊髓损伤、周围神经损伤、糖尿病性神经病变、急慢性缺血性神经病变、神经退行性病变中起着重要的作用。该文就血管内皮生长因子的生物学特点、信号传导及在神经营养与保护方面的近年研究进展作一简要综述。     

少突胶质细胞在脊髓损伤中的病理反应和作用

长期以来的研究认为少突胶质细胞的主要功能是形成中枢神经系统轴突髓鞘、营养和保护轴突,但近年研究发现少突胶质细胞尚有为中枢神经系统提供多种神经营养因子和生长因子、表达轴突生长抑制因子等作用,从而调节神经元、星形胶质细胞,甚至少突胶质细胞本身的生长、发育和存活.少突胶质细胞的凋亡、再生、髓鞘化和形成胶质瘢痕等反应在脊髓损伤继发性病理过程中扮演极其重要的角色,针对少突胶质细胞这一角色制定继发性脊髓损伤治疗新策略,有望成为解决这一医学难题的新切入点.该文就少突胶质细胞的生物学功能及其在脊髓损伤后的反应的研究进展作一综述.     

脐带间充质干细胞治疗脊髓损伤的研究现状

脊髓是神经功能的中介通路,脊髓损伤后,神经元和神经胶质细胞凋亡,脊髓空洞和胶质瘢痕的形成使脊髓功能永久性障碍[1-2].脊髓损伤后神经再生困难主要有如下几个因素:(1)神经细胞的坏死和凋亡,神经轴突的崩解失去传导功能;(2)神经细胞内源性的再生能力有限;(3)损伤局部微环境存在再生抑制因素.     

嗅鞘细胞治疗脊髓损伤的研究进展

脊髓损伤是一种高致残性神经系统损伤性疾病,目前仍然缺乏有效的治疗方法。研究证实嗅鞘细胞是促进脊髓损伤后神经再生的理想种子细胞之一。嗅鞘细胞能通过分泌作用、与星形胶质细胞相互作用、调节炎症反应、迁移特性、髓鞘形成作用、抗氧化作用、脂质调节作用等方式或特性促进轴突的萌发及定向延长,从而发挥神经保护以及神经修复作用。近年来,一些研究利用生物工程、组织工程、重编程等技术从不同方面增强了嗅鞘细胞的疗效,从而为优化脊髓损伤的细胞治疗提供了新的治疗策略。本文将对嗅鞘细胞修复脊髓损伤的机制加以总结,同时归纳近些年优化嗅鞘细胞治疗脊髓损伤策略的研究进展,为进一步开发脊髓损伤后神经修复潜能提供新的研究思路,优化脊髓损伤的细胞治疗效果。     

再生周围神经轴突髓鞘化分子机制研究进展

周围神经损伤后再生轴突髓鞘化是神经再生过程的重要环节。该文从沃勒变性以及细胞外基质、细胞粘附分子、神经调节素等分子生物学角度阐述轴突和髓鞘的内在联系、髓鞘形成的调节等周围神经再生方面的研究进展。对这个重要环节的认识,有助于深入认识神经再生过程,指导髓鞘病变相关疾病的治疗。     

Lithium chloride reinforces the regeneration-promoting effect of chondroitinase ABC on rubrospinal neurons after spinal cord injury

After spinal cord injury, enzymatic digestion of chondroitin sulfate proteoglycans promotes axonal regeneration of central nervous system neurons across the lesion scar. We examined whether chondroitinase ABC (ChABC) promotes the axonal regeneration of rubrospinal tract (RST) neurons following injury to the spinal cord. The effect of a GSK-3beta inhibitor, lithium chloride (LiCl), on the regeneration of axotomized RST neurons was also assessed. Adult rats received a unilateral hemisection at the seventh cervical spinal cord segment (C7). Four weeks after different treatments, regeneration of RST axons across the lesion scar was examined by injection of Fluoro-Gold at spinal segment T2, and locomotor recovery was studied by a test of forelimb usage. Injured RST axons did not regenerate spontaneously after spinal cord injury, and intraperitoneal injection of LiCl alone did not promote the regeneration of RST axons. Administration of ChABC at the lesion site enhanced the regeneration of RST axons by 20%. Combined treatment of LiCl together with ChABC significantly increased the regeneration of RST axons to 42%. Animals receiving combined treatment used both forelimbs together more often than animals that received sham or single treatment. Immunoblotting and immunohistochemical analysis revealed that LiCl induced the expression of inactive GSK-3beta as well as the upregulation of Bcl-2 in injured RST neurons. These results indicate that in vivo, LiCl inhibits GSK-3beta and reinforces the regeneration-promoting function of ChABC through a Bcl-2-dependent mechanism. Combined use of LiCl together with ChABC could be a novel treatment for spinal cord injury.     

干细胞与脊髓损伤的治疗

脊髓损伤的治疗一直是世界性的难题,促进脊髓损伤后神经组织的修复,传统的手术和相应的辅助治疗并未取得突破性进展。组织工程学的发展,为神经组织的修复提供了良好的可能。被移植到患者脊髓损伤区域的干细胞,通过替换受损细胞、减少胶质瘢痕的形成、促进残存神经元细胞轴突再生及突触形成等,可促进脊髓形态及功能恢复。我们就目前应用干细胞治疗脊髓损伤的研究现状进行概述,并对其临床应用前景进行展望。     

Restriction of axonal retraction and promotion of axonal regeneration by chronically injured neurons after intraspinal treatment with glial cell line-derived neurotrophic factor (GDNF)

The response of supraspinal neurons to acute or delayed treatment with GDNF following a spinal cord injury was examined. A cervical level 3 hemisection lesion cavity was created by tissue aspiration in adult, female rats. In one experiment gel foam saturated with GDNF was placed into the lesion cavity immediately after injury to determine if the extent of axonal retraction was affected by neurotrophic factor treatment. One week prior to sacrifice animals received a microinjection of biotinylated dextran amine (BDA) into the red nucleus and reticular formation to label descending spinal pathways by anterograde transport mechanisms. Animals were sacrificed 1 or 4 weeks after injury and treatment with GDNF. The terminal end of injured BDA-labeled rubrospinal and reticulospinal tract axons was identified and the distance from the lesion was measured. In comparison to PBS-treated animals, GDNF-treatment resulted in a significant decrease in the extent of axonal retraction of both rubrospinal and reticulospinal tract axons at 1 week after spinal cord injury for both tracts. At 4 weeks after injury the mean distance from the lesion was less than 240 microm following GDNF-treatment for both tracts, compared to over 480 microm following PBS-treatment. In the second experiment injured supraspinal neurons were labeled by retrograde transport of True Blue that had been placed into the lesion cavity. One month later scar tissue was removed from the cavity by aspiration to enlarge the cavity by approximately 500 microm in a rostral direction. GDNF-saturated gel foam was placed into the cavity for 60 min prior to apposition of an autologous peripheral nerve (PN) graft to the rostral cavity wall. One month later Nuclear Yellow was applied to the distal end of the PN graft and animals were sacrificed after 2 days. The number of supraspinal neurons containing both True Blue and Nuclear Yellow was counted as a measure of axonal regeneration by chronically injured neurons. There was a seven-fold increase in the number of regenerating neurons after GDNF-treatment, with the majority (65%) of dual-labeled neurons located within the reticular formation. These results indicate that GDNF has neuroprotective effects when provided acutely after injury and promotes axonal regeneration when provided in a chronic injury situation.     

Axonal remyelination by cord blood stem cells after spinal cord injury

Human umbilical cord blood stem cells (hUCB) hold great promise for therapeutic repair after spinal cord injury (SCI). Here, we present our preliminary investigations on axonal remyelination of injured spinal cord by transplanted hUCB. Adult male rats were subjected to moderate SCI using NYU Impactor, and hUCB were grafted into the site of injury one week after SCI. Immunohistochemical data provides evidence of differentiation of hUCB into several neural phenotypes including neurons, oligodendrocytes and astrocytes. Ultrastructural analysis of axons reveals that hUCB form morphologically normal appearing myelin sheaths around axons in the injured areas of spinal cord. Colocalization studies prove that oligodendrocytes derived from hUCB secrete neurotrophic hormones neurotrophin-3 (NT3) and brain-derived neurotrophic factor (BDNF). Cord blood stem cells aid in the synthesis of myelin basic protein (MBP) and proteolipid protein (PLP) of myelin in the injured areas, thereby facilitating the process of remyelination. Elevated levels of mRNA expression were observed for NT3, BDNF, MBP and PLP in hUCB-treated rats as revealed by fluorescent in situ hybridization (FISH) analysis. Recovery of hind limb locomotor function was also significantly enhanced in the hUCB-treated rats based on Basso-Beattie-Bresnahan (BBB) scores assessed 14 days after transplantation. These findings demonstrate that hUCB, when transplanted into the spinal cord 7 days after weight-drop injury, survive for at least 2 weeks, differentiate into oligodendrocytes and neurons, and enable improved locomotor function. Therefore, hUCB facilitate functional recovery after moderate SCI and may prove to be a useful therapeutic strategy to repair the injured spinal cord.     

AFGF promotes axonal growth in rat spinal cord organotypic slice co-cultures

This study developed a slice culture model system to study axonal regeneration after spinal cord injury. This model was tested in studies of the roles of acidic fibroblast growth factor (aFGF) and peripheral nerve segments in axonal growth between pieces of spinal cord. Transverse sections of P15-P18 Sprague-Dawley rat spinal cord were collected for organotypic slice cultures. Group I consisted of two slices of spinal cord in contact with each other during the culture period. Group II consisted of two slices that were separated by 3 mm and connected by two segments of intercostal nerves. Group III consisted of single slices for studies of neuron survival. Some cultures from each group included aFGF in the culture medium. Bromodeoxyuridine (BrdU) was included in the medium for some cultures. The results showed three principal findings. First, counts of neurofilament-positive cells demonstrated that treatment with aFGF significantly increased the number of surviving neurons in culture. Second, neurofilament immunostaining and DiI tracing demonstrated axons crossing the junction between the two pieces of spinal cord or growing through the intercostal nerve segments, and these axons were seen only in cultures with aFGF treatment. Third, few cells were double stained for neurofilament and BrdU, and these were found only with aFGF treatment. These results demonstrate that (1) organotypic slice cultures present a useful model to study regeneration from spinal cord injury, (2) aFGF rescues neurons and promotes axonal growth in these cultures, and (3) segments of intercostal nerves promote axon growth between slices of spinal cord.     

Present situation and future aspects of spinal cord regeneration

The central nervous system (CNS) has a limited capacity for regeneration after injury. In spinal cord injury (SCI) patients, total loss of all motor and sensory function occurs below the level of injury. Advances in treatment are expected for orthopedic and spinal surgeons. Recently, evidence of axonal regeneration and functional recovery has been reported in animal spinal cord injury models. Our studies on the roles of inhibitory molecules with a comparison between neonatal and adult animals may help serve as therapeutic targets to enhance axonal regeneration for the injured spinal cord. Also, our cell replacement study indicates the possibility of transplanting neural stem cells to supply the cell source for immature oligodendrocytes, which are thought to be essential for both the myelination and trophic support of regenerating axons in the spinal cord. Administration of neurotrophic factors, prevention of inhibitory factors, and stem cell technology have clinical applications in SCI patients. However, spinal cord regeneration involves a multistep process, and several factors have to be controlled after injury. A combination of several treatments could overcome a nonpermissive environment for spinal cord regeneration. Further understanding of the mechanisms and finding optimal targets of spinal cord regeneration are necessary to obtain successful therapies for SCI patients.Presented at the 76th Annual Meeting of the Japanese Orthopaedic Association, Kanazawa, Japan, May 23, 2003     

神经干细胞移植对脊髓损伤后PLP基因表达的影响

目的：研究神经干细胞(NSCs)移植对大鼠脊髓损伤(SCI)后髓鞘蛋白前脂蛋白(PLP)基因表达的影响，探讨神经干细胞移植促进大鼠SCI后髓鞘再生的机制。方法：NSCs由5-溴-2脱氧尿嘧啶核苷标记法(Brdu)标记。实验分为3组：NSCs移植组(A组)、DMEM填充组(B组)、正常对照组(C组)。大鼠SCI后第7d移植NSCs，应用免疫组化和RT—PCR法观察NSCs移植后是否存活，以及大鼠脊髓损伤区PLP基因表达的动态变化。结果：NSCs移植后在受体脊髓内存活，NSCs移植组较单纯损伤组明显促进了PLP基因在分子和蛋白水平的表达。结论：NSCs移植后可存活并促进PLP基因的表达，是促进脊髓损伤后髓鞘再生的机制之一。     

Long-distance axonal regeneration in the filum terminale of adult rats is regulated by ependymal cells

Studies of regeneration of transected adult central nervous system (CNS) axons are difficult due to lack of appropriate in vivo models. In adult rats, we described filum terminale (FT), a caudal slender extension of the sacral spinal cord and an integral part of the central nervous system (CNS), to use it as a model of spinal cord injury. FT is more than 3 cm long, encompasses a central canal lined with ependymal cells surrounded by a narrow band of axons interspersed with oligodendrocytes and astrocytes but not neurons. Two weeks after the crush of FT, histological, ultrastructural, and axonal tracing studies revealed long distance descending axonal regeneration uniquely in close proximity of the ependymal cells of the central canal. Ependymal cells extended basal processes to form channels encompassing axons apparently regenerating at a rate of more than 2 mm a day. Remarkable increase of axonal sprouting was observed in the sacral spinal cord of Long Evans Shaker (LES) rats with crushed FT. FT offers an excellent model to study mechanisms of axonal regeneration regulated by ependymal cells in the adult CNS.     

Functional recovery after human umbilical cord blood cells transplantation with brain-derived neutrophic factor into the spinal cord injured rat

Summary There have been many efforts to recover neuronal function from spinal cord injuries, but there are some limitations in the treatment of spinal cord injuries.The neural stem cell has been noted for its pluripotency to differentiate into various neural cell types. The human umbilical cord blood cells (HUCBs) are more pluripotent and genetically flexible than bone marrow neural stem cells. The HUCBs could be more frequently used for spinal cord injury treatment in the future.Moderate degree spinal cord injured rats were classified into 3 subgroups, group A: media was injected into the cord injury site, group B: HUCBs were transplanted into the cord injury site, and group C: HUCBs with BDNF (Brain-derived neutrophic factor) were transplanted into the cord injury site. We checked the BBB scores to evaluate the functional recovery in each group at 8 weeks after transplantation. We then, finally checked the neural cell differentiation with double immunofluorescence staining, and we also analyzed the axonal regeneration with retrograde labelling of brain stem neurons by using fluorogold. The HUCBs transplanted group improved, more than the control group at every week after transplantation, and also, the BDNF enabled an improvement of the BBB locomotion scores since the 1 week after its application (P<0.05). 8 weeks after transplantation, the HUCBs with BDNF transplanted group had more greatly improved BBB scores, than the other groups (P<0.001). The transplanted HUCBs were differentiated into various neural cells, which were confirmed by double immunoflorescence staining of BrdU and GFAP & MAP-2 staining. The HUCBs and BDNF each have individual positive effects on axonal regeneration. The HUCBs can differentiate into neural cells and induce motor function improvement in the cord injured rat models. Especially, the BDNF has effectiveness for neurological function improvement due to axonal regeneration in the early cord injury stage. Thus the HUCBs and BDNF have recovery effects of a moderate degree for cord injured rats.     

Chronic regenerative changes in the spinal cord after cord compression injury in rats

Long-term regenerative changes and pathological effects after acute compression injury of the spinal cord were studied in rats. Twenty adult female Wistar rats underwent cord injury by the extradural clip compression technique at T6-7. Following injury, extradural electrodes attached to receiver-stimulators that were implanted subcutaneously were placed proximal and distal to the injury site. The animals were maintained in cages with electromagnetic fields created by encircling antennae. The control animals were in a field adjusted to a frequency below the sensitive frequency range of the receiver-stimulators so that they received no spinal cord stimulation. After 15-20 weeks of continuous spinal cord stimulation, histological sections of the cords were assessed and scored blindly for pathological changes including magnitude and extent of cord injury, and cystic cavitation, and for regenerative changes including proliferation of axons, Schwann cells and ependymal cells, and formation of myelin. In all 20 animals, there was a complete absence of normal cord tissue at the injury site, and cystic cavitation was frequently present at the injury site and beyond. Extensive regenerative changes were seen in all animals including regeneration of axons, Schwann cells and ependymal cells, and formation of myelin. Statistical analysis did not show a significant difference between treatment and control groups.