Endogenous neural progenitor cells in the repair of the injured spinal cord

正Stem cell treatments,and in particular,stem cell transplants have been identified as potential therapeutic strategies for a range of neurodegenerative and acquired conditions of the central nervous system(CNS).Stem cell transplants are seen as a way of replacing lost neurons,or providing a cellular environment that is more permissible for axon and cell regeneration.An alternate strategy     

Cell therapy to repair injured spinal cords: olfactory ensheathing glia transplantation

The absence of spontaneous axonal regeneration in the adult mammalian central nervous system cause devastating functional consequences in patients with spinal cord injuries. During the past decades several attempts have been made in order to find a strategy to repair injured spinal cords in experimental animals, that could provide a novel therapeutic approach in humans. Cell transplantation has been broadly used as an intervention to influence neuronal survival and axonal regeneration in the severed neuraxis. Of the cell types used for transplantation, olfactory ensheathing glia (OEG) promoted a dramatic functional improvement and anatomical repair after complete transection of the adult mammalian spinal cord. These cells can be easily obtained from adult donors opening the possibility of autologous transplantation. Grafting OEG to repair injured spinal cords offers some advantages compared to injections of other cell types. Therefore, OEG have become good candidates to bring about repair in damaged spinal cords. In this article we review OEG transplantation studies, discuss the properties that could account for their axonal growth-promoting ability, and the advantages of using OEG as a repair strategy.     

Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord

The protective effects of erythropoietin on spinal cord injury have not been well described. Here, the eukaryotic expression plasmid pc DNA3.1 human erythropoietin was transfected into rat neural stem cells cultured in vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was injected with non-transfected neural stem cells. Dulbecco's modified Eagle's medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1–4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem cells group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bcl-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythropoietin-neural stem cells group. At 4 weeks, the cavities were clearly smaller and the motor and somatosensory evoked potential latencies were remarkably shorter in the human erythropoietin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythropoietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem cells into the subarachnoid cavity to help repair spinal cord injury and promote the recovery of spinal cord function better than neural stem cell transplantation alone. These findings may lead to significant improvements in the clinical treatment of spinal cord injuries.     

人脐带间充质干细胞移植对大鼠脊髓损伤神经功能恢复的评价

目的 观察人脐带间充质干细胞（human umbilical cordmesenchymal stem cell，hUCMSC）移植对大鼠脊髓损伤神经功能恢复的影响。方法 SD大鼠70只，随机分为3组：脊髓半切＋hUCMSC组（n=30）、脊髓半切＋PBS组（n=30）和假手术组（n=10）。脊髓半切＋hUCMSC组和PBS组又分为头侧注射、尾侧注射和头尾两侧注射三个亚组。移植后1、7、14、21、28d观察大鼠神经功能恢复情况，应用免疫组化检测移植到脊髓的hUCMSC胶质纤维酸性蛋白（GFAP）和神经元特异性烯醇化酶（NSE）表达情况。结果 大鼠脊髓半切损害后，hUCMSC组动物较PBS组有明显的神经功能恢复。植入后28d在宿主脊髓中存活的hUCMSC细胞MABl281（mouse antiuman nuclei monoclonal antibody）染色阳性，免疫组化双标染色显示MABl28l阳性细胞亦分别有NSE或GFAP表达并向损伤部位迁移，hUCMSC来源的GFAP阳性细胞可见明显的树突生长。结论 hUCMSC移植到宿主损伤脊髓后可以存活、向损伤部位迁移，并向神经元样和星形胶质细胞分化，且可促进大鼠脊髓损伤后神经功能恢复。hUCMSC作为一种来源广泛的干细胞用于治疗脊髓损伤可能具有重要的价值。     

Potassium and calcium changes in injured spinal cords

In previous studies, we found large rises in extracellular potassium activity ([K+]e) and falls in extracellular Ca2+ activity ([Ca2+]e) in injured spinal cords. [K+]e recovered rapidly at the impact site within 2 h but ischemia onset at 2-3 h paradoxically did not cause further [K+]e rises. [Ca2+]e, in contrast, remained depressed for long periods of time, suggesting either an effective block of Ca diffusion to the injury site or a deep Ca2+ sink at the impact site. To resolve questions raised by the [K+]e and [Ca2+]e recovery patterns, we used atomic absorption spectroscopy to measure spatial distributions of tissue concentrations of K ([K]t) and Ca ([Ca]t) in cat spinal cords injured by a standardized contusion, compared with uninjured controls. At the impact site, [K]t fell to 51% and 35% of control at 1 and 3 h. The K content of cord surrounding the impact site did not change significantly at 1 h, but K gains in surrounding cord at 3 h approximated K losses from the impact site. The K results indicate that contusion disrupts greater than 80% of cells at the impact site with K loss to adjacent cord, blood, and cerebrospinal fluid. Such losses may explain why subsequent ischemia at the impact site did not cause [K+]e rises. [Ca]t at the impact site increased to 37% and 59% above control at 1 and 3 h. The Ca gain at the impact site exceeded the amount of free extracellular Ca2+ available before injury within 2 cm of the impact site. At 1 h, the Ca lost in cord surrounding the impact site approximated the Ca gain at the impact site. These findings indicate that Ca accumulated at the impact site comes largely from surrounding cord. We propose that Ca sequestration by inorganic phosphates causes a deep Ca sink at the impact site.     

Human umbilical cord blood (HUCB) cells for central nervous system repair

Cellular therapy is a compelling and potential treatment for certain neurological and neurodegenerative diseases as well as a viable treatment for acute injury to the spinal cord and brain. The hematopoietic system offers alternative sources for stem cells compared to those of fetal or embryonic origin. Bone marrow stromal and umbilical cord cells have been used in pre-clinical models of brain injury, directed to differentiate into neural phenotypes, and have been related to functional recovery after engraftment in central nervous system (CNS) injury models. This paper reviews the advantages, utilization and progress of human umbilical cord blood (HUCB) cells in the neural cell transplantation and repair field.     

Application and implications of polyethylene glycol-fusion as a novel technology to repair injured spinal cords

<正>Conventional vs.polyethylene glycol(PEG)-fusion technologies to repair severed spinal axons:Most spinal cord injuries(SCIs)involve cut-or crush-severance of spinal tract axons in the central nervous system(CNS).Clinical outcomes after CNS axonal severance is very poor because proximal segments of CNS axons lack a suitable environment for     

Human umbilical cord blood stem cells upregulate matrix metalloproteinase-2 in rats after spinal cord injury

Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes involved in inflammation, wound healing and other pathological processes after neurological disorders. MMP-2 promotes functional recovery after spinal cord injury (SCI) by regulating the formation of a glial scar. In the present study, we aimed to investigate the expression and/or activity of several MMPs, after SCI and human umbilical cord blood mesenchymal stem cell (hUCB) treatment in rats with a special emphasis on MMP-2. Treatment with hUCB after SCI altered the expression of several MMPs in rats. MMP-2 is upregulated after hUCB treatment in spinal cord injured rats and in spinal neurons injured either with staurosporine or hydrogen peroxide. Further, hUCB induced upregulation of MMP-2 reduced formation of the glial scar at the site of injury along with reduced immunoreactivity to chondroitin sulfate proteoglycans. Blockade of MMP-2 activity in hUCB cocultured injured spinal neurons reduced the protection offered by hUCB which indicated the involvement of MMP-2 in the neuroprotection offered by hUCB. Based on these results, we conclude that hUCB treatment after SCI upregulates MMP-2 levels and reduces the formation of the glial scar thereby creating an environment suitable for endogenous repair mechanisms.     

Reflex changes induced by clonidine in spinal cord injured patients

In a single blind study of 6 spinal cord injured (SCI) men, the effects of clonidine, a selective alpha-2 adrenergic agonist, on spasticity were compared to diazepam and placebo. Since a potential side-effect of clonidine is postural hypotension, a combination of clonidine and desipramine was also tested. Vibration of the leg will inhibit the H reflex in a normal subject; whereas, this inhibition is markedly reduced in SCI patients with spasticity. A vibratory inhibition index (VII) was derived for each treatment. The pre-treatment VII was 92.08 +/- 3.15%; for SCI subjects, compared to 46.5 +/- 7.7% for 6 normal subjects. Clonidine significantly reduced the mean index of SCI patients to 59.42 +/- 3.91% (p less than 0.001). The VII for placebo, diazepam and the clonidine-desipramine combination were not statistically different than the pre-treatment values in SCI patients. In conclusion, clonidine has an anti-spasticity effect in SCI patients, both subjectively, and objectively, in terms of vibratory inhibition of the H reflex.     

Bridging the injured spinal cord with neural stem cells

<正>Spinal cord injury(SCI)damages not only the gray matter neurons,but also the white matter axonal tracts that carry signals to and from the brain,resulting in permanent loss of function below injury.Neural stem cells(NSCs)have high therapeutic potential for reconstruction of the injured spinal cord since they can potentially form neuronal relays to bridge functional connectivity between separated spinal cord segments.This requires host axonal regeneration into and connectivity with donor neurons,and axonal growth and connectivity of donor neurons to host central nervous system(CNS)circuitry.In this mini-review,we will discuss key studies that explore novel neuronal relay formation by grafting NSCs in models of SCI,with emphasis on long-distance axonal growth and connectivity of NSCs grafted into injured spinal cord.     

他克莫司促进神经干细胞移植大鼠脊髓损伤的再生与修复

背景：研究证实,他克莫司不仅抑制T细胞的增殖、活化,还能抑制小胶质细胞、巨噬细胞等炎症细胞在损伤局部聚集、活化及相关炎症因子的释放,减轻继发性炎症反应对原发损伤周围正常组织的破坏,从而对损伤局部的神经组织起保护作用。 目的：观察他克莫司对神经干细胞移植大鼠脊髓损伤后再生修复的影响。 方法：分离培养孕13d SD大鼠神经干细胞。显微镜下动脉瘤夹夹闭SD大鼠T8脊髓,建立压迫型脊髓损伤动物模型。损伤后7 d随机数字表分为3组：对照组,于损伤中心定向注射生理盐水;细胞移植组,于损伤中心定向注射神经干细胞;他克莫司组,于损伤中心定向注射神经干细胞同时给予免疫抑制剂他克莫司1 mg/(kg•d)腹腔注射连续7 d。1,2,4,8周后,通过BDA顺行示踪、苏木精-伊红与免疫组化染色及电镜检测,观察移植后脊髓组织再生和神经元的变化。 结果与结论：对照组在损伤中心端远侧无神经纤维通过。细胞移植组与他克莫司组在治疗1周后有部分神经纤维通过,8周均有部分BDA阳性标记的皮质脊髓束再生通过脊髓损伤部位,特别是他克莫司组可延续至距损伤中心1.7 cm 。苏木精-伊红染色显示,细胞移植组与他克莫司组2周时坏死灶开始缩小,泡沫细胞减少。电镜结果显示,他克莫司组1周时即出现较正常的微丝和微管结构,8周时星形细胞、许旺细胞、髓鞘典型多见,神经轴突的终末有较多的兴奋性递质和不典型的轴树连接,出现较多的结构正常的髓鞘。说明损伤大鼠移植神经干细胞后联合应用他克莫司后可减轻早期的急性炎症反应,保证神经细胞的存活,具有神经保护和神经营养作用,可加快神经功能的恢复。     

Genetically modified human umbilical cord blood cells as a promising strategy for treatment of spinal cord injury

正Spinal cord injury(SCI)continues to be a pressing health and social problem.The injury leads to neuronal and glial cell death accompanied by degeneration of nerve fibers.There are currently no particularly effective treatments.SCI causes profound disability of people affected and has attracted increased attention in the international field of neuroregeneration.For the past two decades,     

脐血贴壁层细胞和骨髓基质细胞对脐血单个核细胞增殖能力的影响★

目的：比较脐血贴壁层细胞和骨髓的基质细胞对脐血单个核细胞增殖能力的影响。 方法：实验于2005-02/07在吉林省肿瘤防治研究所完成。①对象：正常足月顺产儿脐带由长春市妇产医院提供；肋骨取自吉林省肿瘤医院外科手术患者，排除血液疾患。产妇及外科手术患者对治疗及实验均签署知情同意书，实验经医院医学伦理委员会批准。②实验方法：采用红细胞裂解法制备有核细胞，以Ficoll-paque淋巴细胞分层液按密度梯度离心法分离出脐血单个核细胞，体外培养24 h后去除悬浮细胞，收集脐血贴壁层细胞，加入到24孔板中，2×106 L-1/孔，再加入含12.5%马血清、12.5%胎牛血清、1×10-7 mol/L氢化考地松的IMDM培养体系1 mL。将肋骨剪成3 cm小块，冲洗髓腔收集细胞悬液，用淋巴细胞分层液分离出骨髓单个核细胞，同法进行骨髓基质细胞的培养。分别将两种细胞于37 ℃、体积分数为0.05的CO2条件下培养3周后，各自加入到24孔板，2×106 L-1/孔，同时每孔再加入1×106 L-1脐血单个核细胞，培养7 d。以未添加脐血贴壁层细胞及骨髓基质细胞的脐血单个核细胞作为空白对照。③实验评估：倒置显微镜下观察脐血贴壁层细胞与骨髓基质细胞的生长状态。采用流式细胞仪测定脐血单个核细胞周期分布。甲基纤维素半固体培养法测定脐血单个核细胞混合集落形成单位情况。 结果：①脐血贴壁层细胞与骨髓基质细胞的形态：培养2周时，多数脐血贴壁层细胞呈大小不等的圆形、椭圆形，少部分呈不规则形；骨髓基质细胞主要为梭形，细胞排列成漩涡状、辐射状或相互缠绕成束状。②细胞周期：与空白对照相比，经脐血贴壁层细胞、骨髓基质细胞干预的脐血单个核细胞S+G2+M期所占比例均显著升高（P < 0.05）。③混合集落形成单位：经脐血贴壁层细胞、骨髓基质细胞干预后的脐血单个核细胞，其混合集落形成单位的数量显著高于空白对照（P < 0.05）。 结论：①脐血贴壁层细胞与骨髓基质细胞在形态上具有一定差异，但均能提高脐血单个核细胞体外增殖能力。②脐血贴壁层细胞可替代骨髓基质细胞作为脐血造血细胞体外扩增的辅助条件。     

Use of human umbilical cord blood (HUCB) cells to repair the damaged brain

Neurodegenerative diseases as well as acute center nervous system (CNS) injuries remains a problematic and frustrating area of medicine in terms of treatments and cures, which is mostly due to the complex circuitry of the CNS along with our limited knowledge. Therapeutically, the last two and a half decades have offered new hope for those suffering from neurodegenerative diseases or injuries with advent of new drug discoveries and cellular therapies. Cell transplantation is a compelling and potential treatment for certain neurological and neurodegenerative diseases as well as for acute injuries to the spinal cord and brain. The hematopoietic system offers an alternative source of cells that is easily obtainable, abundant, and reliable when compared to cells obtained from fetal or embryonic origins. Human umbilical cord blood (HUCB) cells have been used clinically for over ten years to treat both malignant and non-malignant diseases. With in the last five years these cells have been used pre-clinically in animal models of brain and spinal cord injuries, in which functional recovery have been shown. This paper reviews the advantages, utilization, and progress of HUCB cells in the field of cellular transplantation and repair.     

嗅鞘细胞移植损伤脊髓组织的超微结构变化

背景：脊髓损伤后神经功能难以自行恢复,嗅鞘细胞具有外周性和中枢性两种胶质细胞的成鞘功能,是修复受损神经最有前途的种子细胞。嗅鞘细胞移植到受损脊髓后的组织学和超微结构的变化可能帮助解释嗅鞘细胞发挥修复作用的机制。 目的: 验证嗅球源性嗅鞘细胞移植对脊髓损伤功能恢复的促进作用,并观察移植的嗅鞘细胞对神经元和轴突组织和超微结构的影响。 方法：将已制备脊髓模型的Wistar大鼠随机分为3组,对照组不做任何注射操作,DMEM/F12组注射DMEM/F12培养基,嗅鞘细胞组注射嗅鞘细胞悬液。每周进行肢体活动BBB评分,8周后取脊髓标本进行组织学和免疫组织化学观察,评价脊髓损伤的修复情况,并观察嗅鞘细胞移植对脊髓组织和超微结构的影响。 结果与结论：3组动物均出现后肢运动功能的恢复,嗅鞘细胞组优于对照组和DMEM/F12组,在4周后更为明显。组织学观察可见,在嗅鞘细胞组可见有神经纤维通过损伤处。损伤处附近,嗅鞘细胞组脊髓腹侧的神经纤维和神经元形态较好,损伤较轻。而对照组和DMEM/F12组神经纤维和神经元损害严重。嗅鞘细胞组的caspsase-3阳性细胞数少于对照组和DMEM/F12组。超微结构观察可见,嗅鞘细胞组的神经纤维和细胞形态均优于对照组和DMEM/F12组。结果表明嗅鞘细胞移植对大鼠脊髓损伤修复有明显的促进作用,并可恢复损伤神经的部分功能,对受损神经纤维和神经元有保护作用。     

香丹注射液诱导人脐血间充质干细胞向神经细胞的分化

背景：选择一种高效、细胞损伤低的诱导人脐血间充质干细胞分化为神经细胞的方法是其应用于临床的前提。 目的：拟采用传统中药香丹注射液联合生长因子诱导脐血间充质干细胞向神经细胞方向分化,并且与单纯生长因子的诱导作用进行比较。 设计、时间及地点：免疫细胞化学水平的细胞观察实验,于2006-09/2008-04在潍坊医学院组织胚胎学教研室完成。 材料：人脐血标本取自潍坊市妇幼保健院,香丹注射液的有效成分为丹参素。 方法：采用密度梯度离心法分离人脐血单个核细胞,贴壁法筛选出人脐血间充质干细胞,体外扩增,以免疫荧光方法检测表面抗原。分为2组进行诱导分化,香丹注射液联合生长因子诱导组采用30 g/L香丹注射液联合表皮生长因子和碱性成纤维细胞生长因子诱导脐血间充质干细胞向神经细胞方向分化,并且与单纯生长因子诱导组进行比较。 主要观察指标：采用免疫细胞化学方法和免疫荧光方法检测诱导前后神经元特异性标志（神经元核抗原、β-TubulinⅢ）和神经胶质纤维酸性蛋白的表达。 结果：香丹注射液联合生长因子诱导法对脐血间充质干细胞损伤作用小,诱导效率高。脐血间充质干细胞经香丹注射液诱导后出现类似神经细胞的形态改变,胞体呈椭圆形,伸出长突起。免疫组织化学和免疫荧光方法鉴定显示,诱导后的细胞能特异性表达神经元核抗原和β-TubulinⅢ,而神经胶质纤维酸性蛋白阳性细胞较少。香丹注射液联合生长因子诱导组的 β-TubulinⅢ和神经元核抗原的阳性细胞率显著高于生长因子诱导组（P < 0.05）,神经胶质纤维酸性蛋白阳性细胞率明显低于生长因子诱导组（P < 0.05）。 结论：香丹注射液联合生长因子诱导对脐血间充质干细胞损伤作用小,诱导效率高,优于生长因子诱导法。且体外诱导后细胞主要分化为神经元样细胞,而非星形胶质细胞。     

Novel cellular approaches to repair of neurodegenerative disease: from Sertoli cells to umbilical cord blood stem cells

Neural transplantation is a promising approach to the treatment of neurodegenerative diseases and brain injury that has been shown to be efficacious in many animal models. However, the use of fetal tissue limits the acceptability and widespread application of this technique. In this review we discuss possible alternatives cell sources that may be used to repair the brain and spinal cord, with a focus on Sertoli cells, hNT Neurons, bone marrow and umbilical cord blood derived stem cells.