大鼠胚胎神经干细胞移植治疗脑出血的实验研究

目的 研究大鼠胚胎神经干细胞移植治疗脑出血的可行性。方法 从孕龄16天的大鼠胚胎脑组织中分离、培养神经干细胞并诱导其分化，通过免疫组化学技术研究其特性。制作大鼠脑出血模型，3天后将未分化的神经干细胞注入血肿同侧或对侧的尾状核内，记录损伤和移植后的大鼠运动功能。不同时间杀死大鼠，研究移植后的干细胞在体内分化和迁徙的情况。结果 实验中分离、培养的神经干细胞体外能够被诱导分化成神经元、光突胶质细胞和星形胶质细胞，血肿同侧移植干细胞的大鼠运动功能的改善显著好于血肿对侧移植干细胞组及未移植干细胞的对照组。免疫组化方法证实移植后的干细胞在体内可分化成神经元和胶质细胞，并向损伤区域迁徙。结论 大鼠胚胎神经干细胞体内、体外均具有多向分化潜能，其分化成各种类型神经细胞的比例与所处的外界环境有关，在脑内靠近损伤部位移植胚胎神经干细胞后能够有效改善脑出血动物的运动功能。     

神经干细胞植入阿尔茨海默病鼠脑后的效果观察

神经干细胞移植治疗阿尔茨海默病鼠包括细胞替代治疗和基因治疗,神经干细胞移植阿尔茨海默病鼠脑后其组织形态学与行为学效应均可以得到不同程度的修复和改善。细胞替代治疗中,神经干细胞与神经营养因子联合移植效应优于单纯的神经干细胞移植,但目前对神经干细胞体内分化机制的不确定导致了神经干细胞移植治疗的盲目性,同时对影响其疗效的各种可能因素也缺乏比较研究。神经干细胞基因治疗具有细胞替代和基因治疗的双重作用,但尚处于研究的初期阶段,仍以NGF,BDNF,GDNF等单一营养因子基因修饰的神经干细胞移植治疗为主,且转基因神经干细胞移植入阿尔茨海默病鼠脑后外源基因表达效率、促分化、功能修复情况以及安全性的研究还很缺乏。目前神经干细胞移植治疗阿尔茨海默病鼠脑后的疗效检测技术手段比较单一,免疫组化方法与活体示踪技术的结合、形态学指标与功能学指标的综合检测是疗效检测的发展趋势。     

神经干细胞移植对脑出血模型大鼠神经功能的影响☆

背景：外源性神经干细胞具有神经修复作用，可能对脑出血后的神经功能恢复起到一定的作用。 目的：观察胎鼠神经干细胞的体外生长、分化及移植到脑出血大鼠后的存活、迁徙、分化情况，探讨神经干细胞对脑出血模型大鼠受损神经功能的修复作用。 设计：完全随机分组设计，对照动物实验。 单位：复旦大学附属华山医院神经外科 材料：选用健康雄性成年SD大鼠18只为受体，体质量280~320 g，由中国科学院上海实验动物中心提供。实验用鼠抗BrdU为Neomarkers产品, 鼠抗胶质纤维酸性蛋白和兔抗微管相关蛋白2 为Chemicon产品。 方法：实验于2006-02/12在复旦大学附属上海医学院解剖组胚实验室完成。从胎龄14 d的胎鼠海马中分离、培养、鉴定神经干细胞。16只受体SD大鼠被随机分为3组：对照组，PBS组和移植神经干细胞组。均通过尾状核内注射自体动脉血制作大鼠脑出血模型。移植NSC组在造模后30 min在血肿腔周围四点分别移植浓度为2×1011 L-1神经干细胞悬液5μL；PBS组于相同时间点在脑内相同部位注射PBS；PBS和神经干细胞的移植方法同自体血的移植方法。对照组大鼠在造模后30 min只造成四点损伤，不注射任何物质。 主要观察指标：在造模后立即，1，3，5，14，21，28 d采用前肢评分和转身评分对大鼠神经功能进行评估。大鼠于造模后28 d麻醉后取脑，并通过双标胶质纤维酸性蛋白、微管相关蛋白2、BrdU免疫组化来检测移植入脑的神经干细胞在体内的分化情况。 结果：①神经功能评分：造模后5 d，各组差异无显著性意义（P > 0.05）。造模后14~28 d，干细胞移植组较其他3组明显改善（P < 0.05）。②脑组织切片双免疫组织学双标染色结果：干细胞移植组血肿周围凋亡细胞少于PBS组。受体大鼠脑组织切片显示有BrdU, 微管相关蛋白2,胶质纤维酸性蛋白阳性细胞，说明神经干细胞可以在宿主脑内存活、迁徙和分化，可以分化为神经元样细胞和神经胶质样细胞。 结论：神经干细胞移植可能通过分化为神经元样细胞和神经胶质细胞促进大鼠脑出血的神经功能恢复。     

早期分化的神经干细胞移植治疗大鼠脑梗死的实验研究

目的研究早期分化的神经干细胞移植治疗脑梗死的可能性。方法从Wistar新生大鼠的大脑分离培养神经干细胞,取传代的神经干细胞诱导分化并经BrdU(5-溴脱氧尿嘧啶)标记后移植到脑梗死对侧的侧脑室,移植后对大鼠的功能恢复进行评价,用免疫组织化学方法鉴定移植的细胞在脑内的迁移和分化情况。结果将早期分化的神经干细胞移植到鼠脑后2周在梗死灶对侧可发现移植的细胞,4周时移植的细胞在梗死灶内分化为神经细胞,大鼠的学习功能和神经功能恢复较对照组均有明显改善。结论早期分化的神经干细胞移植到大鼠脑内仍能存活,并能有效穿过脑脊液———脑屏障迁移到脑梗死的部位;且分化为神经细胞。     

神经干细胞移植促进脑出血大鼠功能恢复

目的:观察胎鼠神经干细胞体外的生长、分化及移植到脑出血大鼠后的存活、迁徙、分化情况。研究神经干细胞对脑出血后神经功能损害的可能修复作用。方法:通过尾状核内注射自体动脉血制作大鼠脑出血模型。分离、培养大鼠胎鼠神经干细胞并移植于成年大鼠尾状核,对大鼠脑出血后的神经恢复情况进行功能评价。结果:神经干细胞可以在宿主脑内存活、迁徙和分化,神经干细胞移植组大鼠运动功能较对照组明显改善。结论:神经干细胞移植能促进改善大鼠脑出血的神经功能恢复。     

脑出血大鼠脑内神经干细胞移植的研究

目的分离并克隆新生大鼠神经干细胞,研究其移植入脑出血大鼠脑内的生物学特征,了解神经干细胞移植治疗脑出血的可行性.方法用尾状核注射Ⅶ型胶原酶制作脑出血模型,从Wistar新生大鼠脑室下区分离并克隆神经干细胞,经Brdu(5-溴脱氧尿嘧啶)掺入标记后移植入脑出血同侧的侧脑室或脑出血对侧的尾状核中.经免疫组织化学鉴定了解移植细胞在大鼠脑内的生存、迁移及分化情况.结果将稳定培养的神经干细胞移植入脑出血大鼠脑内,发现移植后4 d移植细胞仍存在.侧脑室移植组中移植细胞多在侧脑室周边区存在,尾状核移植组可见移植细胞开始向对侧迁移.免疫荧光双标证实细胞大多分化成神经元,少部分分化成胶质细胞.结论神经干细胞移植入脑出血大鼠脑内后能够存活,并能有效地穿过室管膜和向脑出血部位迁移.移植细胞在脑内大部分分化成神经元,少部分分化成胶质细胞.     

神经干细胞移植治疗脊髓损伤的研究现状

脊髓完全横断性损伤引起的永久性神经功能障碍的治疗，目前还没有很有效的方法。文章介绍了对神经干细胞培养、神经干细胞表型控制及神经干细胞移植等方法的分析，介绍了神经干细胞移植治疗脊髓损伤的研究现状。目前神经干细胞移植的动物实验主要致力于提高轴突再生能力、替代细胞成分、阻止脱髓鞘和使髓鞘再生等方面，具有促进感觉及运动功能恢复的客观结果，有些甚至已经进入了临床实验阶段。不过神经干细胞的成功应用还受到很多因素的影响，诸如移植剂量、细胞生长因子的活力，细胞移植的风险等，尤其是其效果还需要进一步研究、评价，并且需要长时间的随访。 关键词：神经干细胞；脊髓损伤；细胞移植     

神经干细胞移植治疗小儿脑性瘫痪及所存在的问题

脑性瘫痪是儿童时期最常见且终生存在的运动性残疾,目前尚无有效治疗手段,细胞移植和基因治疗技术的飞速发展为治疗此类疾病带来了希望。近两年国内外出现有关神经干细胞移植治疗脑性瘫痪的报道,这给脑性瘫痪的治疗研究提供了新的思路。文章阐述了小儿脑性瘫痪的神经干细胞机制,并回顾近年来国内外关于神经干细胞移植在动物实验及临床中的有效应用,为神经干细胞治疗小儿脑性瘫痪的可行性提供依据。但神经干细胞移植治疗脑性瘫痪还存在一些问题：移植仍然存在免疫排斥反应;如何促进神经干细胞的快速增殖;如何实现神经干细胞的定向诱导分化;如何评价神经干细胞移植以后患儿的改善情况,移植时机,移植量,移植部位,移植方式等一系列问题均有待于基础和临床学科的共同研究和探讨。     

神经干细胞与脊髓损伤的治疗*★

学术背景：脊髓损伤患者损伤平面以下感觉、运动、反射及尿便功能障碍，胚胎的脊髓组织、许旺细胞以及基因修饰的功能细胞等载体移植均有助于神经系统的恢复。其中神经干细胞因与损伤区域的细胞同源，故具有独特的治疗优势。 目的：深入认识神经干细胞与脊髓损伤治疗领域的相关研究进展。 检索策略：由该论文的研究人员应用计算机检索Pubmed与Science direct数据库2000-01/2007-05的相关文献，检索词“neural stem cells，spinal cord injury”，并限定文章语言种类为English。共检索到132篇文献，对资料进行初审，纳入标准：①文章所述内容应与神经干细胞治疗脊髓损伤相关。②同一领域选择近期发表或在权威杂志上发表的文章。③近4年文献。排除标准：①重复性研究。②Meta分析。③综述文献。 文献评价：文献的来源主要是通过对神经干细胞治疗脊髓损伤方面内容进行汇总分析，所选用的23篇文献均为临床或基础实验研究。 资料综合：①脊髓损伤后，血-脊髓屏障被破坏，多种炎性因子进入损伤区域，触发细胞坏死和凋亡等级联效应。从病理生理机制角度，脊髓损伤后出现的局部微环境改变也是造成神经系统再生失败的重要原因。②神经干细胞具备自我维持和更新的能力、增殖分裂能力、自我更新能力、多向分化潜能、一定的迁移能力。在哺乳动物中枢神经系统内，神经干细胞可从胚胎、胎儿和成人脑与脊髓组织的不同部位分离出来，并能够在体外或体内扩增，分化为神经元、星形胶质细胞和少突胶质细胞。③将神经干细胞或其分化产物移植至宿主脊髓内，继而分化为相应的神经细胞是多种中枢神经系统疾病治疗的共同途径。目前仍有许多问题亟待解决，如神经干细胞定向分化的诱导信号通路及相关作用机制；体外操作对神经干细胞分化特性的影响；移植后神经干细胞的功能状态及迁移、分化调控等。 结论：利用神经干细胞进行细胞替代和转基因治疗脊髓损伤具有较好的临床应用前景。     

干细胞及组织移植治疗甲状腺功能减退症

背景：干细胞及甲状腺组织移植治疗甲状腺功能减退症已经取得了一定的成果。 目的：回顾分析干细胞与组织移植治疗甲状腺功能减退症的基础和临床研究进展。 方法：由作者检索1980/2010 PubMed 数据库及万方数据库有关干细胞与甲状腺移植治疗甲状腺功能减退症的基础和临床研究。 结果与结论：自体甲状腺组织移植治疗不可逆性甲状腺功能减退症的作用是肯定的,但存在不足之处,比如需要移植多少甲状腺组织才能使甲状腺功能保持在正常状态,移植后的长期效果还需要更多样本、更长期的跟踪随访来评价。胚胎干细胞在体外环境下可诱导分化为甲状腺细胞,但受伦理学和细胞移植排斥反应等原因困扰。脐血间充质干细胞具有来源丰富、易于采集、保存和运输、无异体排斥、避免伦理争议等诸多优点,在不同诱导条件下能够向不同谱系分化,已被广泛用于治疗各种疾病,但能否通过多种途径诱导其分化为甲状腺细胞,并通过移植方法来治疗甲状腺功能减退症仍需深入研究。     

Neural induction of adult bone marrow and umbilical cord stem cells

Recent reports of neural differentiation of postnatally derived bone marrow and umbilical cord cells have transformed our understanding of the biology of cell lineages, differentiation, and plasticity. While much controversy remains, it is clear that adult tissues, and bone marrow in particular, are composed in part of cells with much more diverse lineage capacity than previously thought. Traditionally, cell-based therapies for the CNS have been derived from fetal or embryonic origin. By harnessing the neural potential of readily-available and accessible adult bone marrow and umbilical cord blood stem cells, substantial ethical and technical dilemmas may be circumvented. This review will focus on the potential of adult bone marrow derived cells and umbilical cord blood stem cells for cell replacement and repair therapies of the central nervous system. The various isolation protocols, phenotypic properties, and methods for in vivo and in vitro neural differentiation of mesenchymal stem cells/marrow stromal cells (MSC), hematopoietic stem cells (HSC), multipotent adult progenitor cells (MAPCs), and umbilical cord blood stem cells (UCBSC) will be discussed. Current progress regarding transplant paradigms in various disease models as well as in our understanding of transdifferentiation mechanisms will be presented.     

Human neural stem cells genetically modified for brain repair in neurological disorders

Existence of multipotent neural stem cells (NSC) has been known in developing or adult mammalian CNS, including humans. NSC have the capacity to grow indefinitely and have multipotent potential to differentiate into three major cell types of CNS, neurons, astrocytes and oligodendrocytes. Stable clonal lines of human NSC have recently been generated from the human fetal telencephalon using a retroviral vector encoding v‐myc. One of the NSC lines, HB1.F3, carries normal human karyotype of 46XX and has the ability to self‐renew, differentiate into cells of neuronal and glial lineages, and integrate into the damaged CNS loci upon transplantation into the brain of animal models of Parkinson disease, HD, stroke and mucopolysaccharidosis. F3 human NSC were genetically engineered to produce L‐dihydroxyphenylalanine (L‐DOPA) by double transfection with cDNA for tyrosine hydroxylase and guanosine triphosphate cylohydrolase‐1, and transplantation of these cells in the brain of Parkinson disease model rats led to L‐DOPA production and functional recovery. Proactively transplanted F3 human NSC in rat striatum, supported the survival of host striatal neurons against neuronal injury caused by 3‐nitropro‐pionic acid in rat model of HD. Intravenously introduced through the tail vein, F3 human NSC were found to migrate into ischemic lesion sites, differentiate into neurons and glial cells, and improve functional deficits in rat stroke models. These results indicate that human NSC should be an ideal vehicle for cell replacement and gene transfer therapy for patients with neurological diseases. In addition to immortalized human NSC, immortalized human bone marrow mesenchymal stem cell lines have been generated from human embryonic bone marrow tissues with retroviral vectors encording v‐myc or teromerase gene. These immortalized cell lines of human bone marrow mesenchymal stem cells differentiated into neurons/glial cells, bone, cartilage and adipose tissue when they were grown in selective inducing media. There is further need for investigation into the neurogenic potential of the human bone marrow stem cell lines and their utility in animal models of neurological diseases.     

Stem cell‐based cell therapy for Huntington disease: A review

Huntington disease (HD) is a devastating neurodegenerative disorder and no proven medical therapy is currently available to mitigate its clinical manifestations. Although fetal neural transplantation has been tried in both preclinical and clinical investigations, the efficacy is not satisfactory. With the recent explosive progress of stem cell biology, application of stem cell‐based therapy in HD is an exciting prospect. Three kinds of stem cells, embryonic stem cells, bone marrow mesenchymal stem cells and neural stem cells, have previously been utilized in cell therapy in animal models of neurological disorders. However, neural stem cells were preferably used by investigators in experimental HD studies, since they have a clear capacity to become neurons or glial cells after intracerebral or intravenous transplantation, and they induce functional recovery. In this review, we summarize the current state of cell therapy utilizing stem cells in experimental HD animal models, and discuss the future considerations for developing new therapeutic strategies using neural stem cells.     

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.     

骨髓间充质干细胞治疗脑出血后神经功能障碍的研究进展

本文目的是对骨髓间充质干细胞治疗脑出血后功能障碍的研究进展进行综述，为脑出血的治疗提供参考。脑出血是一种对脑组织损害严重的疾病，在全世界范围内造成了沉重的公共健康负担。该病具有高致残率、高死亡率的特点，且治疗效果欠佳。骨髓间充质干细胞在移植后具有能定向分化为神经元并旁分泌神经营养因子的功能，成为治疗脑出血的新选择。     

Bone marrow mesenchymal stem cells repair spinal cord ischemia/reperfusion injury by promoting axonal growth and anti-autophagy

Bone marrow mesenchymal stem cells can differentiate into neurons and astrocytes after trans- plantation in the spinal cord of rats with ischemia/reperfusion injury. Although bone marrow mesenchymal stem cells are known to protect against spinal cord ischemia/reperfusion injury through anti-apoptotic effects, the precise mechanisms remain unclear. In the present study, bone marrow mesenchymal stem cells were cultured and proliferated, then transplanted into rats with ischemia/reperfusion injury via retro-orbital injection. Immunohistochemistry and immunofluorescence with subsequent quantification revealed that the expression of the axonal regeneration marker, growth associated protein-43, and the neuronal marker, microtubule-as- sociated protein 2, significantly increased in rats with bone marrow mesenchymal stem cell transplantation compared with those in rats with spinal cord ischemia/reperfusion injury. Fur- thermore, the expression of the autophagy marker, microtubule-associated protein light chain 3B, and Beclin 1, was significantly reduced in rats with the bone marrow mesenchymal stem cell transplantation compared with those in rats with spinal cord ischemia/reperfusion injury. Western blot analysis showed that the expression of growth associated protein-43 and neuro- filament-H increased but light chain 3B and Beclin 1 decreased in rats with the bone marrow mesenchymal stem cell transplantation. Our results therefore suggest that bone marrow mes- enchymal stem cell transplantation promotes neurite growth and regeneration and prevents autophagy. These responses may likely be mechanisms underlying the protective effect of bone marrow mesenchymal stem cells against spinal cord ischemia/reperfusion injury.     

660 nm red light-enhanced bone marrow mesenchymal stem cell transplantation for hypoxic-ischemic brain damage treatment

Bone marrow mesenchymal stem cell transplantation is an effective treatment for neonatal hypoxic-ischemic brain damage. However, the in vivo transplantation effects are poor and their survival, colonization and differentiation efficiencies are relatively low. Red or near-infrared light from 600–1,000 nm promotes cellular migration and prevents apoptosis. Thus, we hypothesized that the combination of red light with bone marrow mesenchymal stem cell transplantation would be effective for the treatment of hypoxic-ischemic brain damage. In this study, the migration and colonization of cultured bone marrow mesenchymal stem cells on primary neurons after oxygen-glucose deprivation were detected using Transwell assay. The results showed that, after a 40-hour irradiation under red light-emitting diodes at 660 nm and 60 mW/cm2, an increasing number of green fluorescence-labeled bone marrow mesenchymal stem cells migrated towards hypoxic-ischemic damaged primary neurons. Meanwhile, neonatal rats with hypoxic-ischemic brain damage were given an intraperitoneal injection of 1 × 106 bone marrow mesenchymal stem cells, followed by irradiation under red light-emitting diodes at 660 nm and 60 mW/cm2 for 7 successive days. Shuttle box test results showed that, after phototherapy and bone marrow mesenchymal stem cell transplantation, the active avoidance response rate of hypoxic-ischemic brain damage rats was significantly increased, which was higher than that after bone marrow mesenchymal stem cell transplantation alone. Experimental findings indicate that 660 nm red light emitting diode irradiation promotes the migration of bone marrow mesenchymal stem cells, thereby enhancing the contribution of cell transplantation in the treatment of hypoxic-ischemic brain damage.     

Neural cells derived from adult bone marrow and umbilical cord blood

Under experimental conditions, tissue-specific stem cells have been shown to give rise to cell lineages not normally found in the organ or tissue of residence. Neural stem cells from fetal brain have been shown to give rise to blood cell lines and conversely, bone marrow stromal cells have been reported to generate skeletal and cardiac muscle, oval hepatocytes, as well as glia and neuron-like cells. This article reviews studies in which cells from postnatal bone marrow or umbilical cord blood were induced to proliferate and differentiate into glia and neurons, cellular lineages that are not their normal destiny. The review encompasses in vitro and in vivo studies with focus on experimental variables, such as the source and characterization of cells, cell-tracking methods, and markers of neural differentiation. The existence of stem/progenitor cells with previously unappreciated proliferation and differentiation potential in postnatal bone marrow and in umbilical cord blood opens up the possibility of using stem cells found in these tissues to treat degenerative, post-traumatic and hereditary diseases of the central nervous system.     

脐带血干细胞的基础与临床应用进展

脐带血含有丰富的造血干细胞，已经成为替代骨髓移植治疗多种疾病的良好资源，但尚存多种不足与难题。国内外大量研究证实, 脐带血干细胞具有肯定的支持造血功能，在体外培养条件下可以扩增，并且在一定的诱导作用下可分化为骨、软骨、肝、内皮等多种组织细胞。脐带血干细胞具有低免疫原性等很多优点，已成为干细胞研究领域的热点。目前分离培养脐带血干细胞的方法多样，日趋成熟，临床应用脐带血干细胞移植治疗的疾病已达80余种，但仍存在许多弊端, 尚需进一步的研究。     

干细胞在脑性瘫痪治疗中的应用

背景：干细胞在适当条件下可以分化为神经元细胞、星形胶质细胞与少突胶质细胞，可能从根本上改善脑性瘫痪患儿神经元缺失及神经胶质细胞变性，进而改善患儿脑功能障碍，从理论上达到根治目的。 目的：回顾性分析不同来源干细胞经不同途径治疗脑性瘫痪患儿的疗效。 方法：由第一作者检索1992/2011 PubMed数据及万方数据库有关脑性瘫痪的治疗及不同来源干细胞在治疗脑性瘫痪等方面的文献。 结果与结论：神经干细胞在动物神经功能损伤中的修复作用已有很多国内外报道，但目前其在人体的临床应用仍处于临床试验阶段。虽然胚胎、骨髓血、胎儿脐带血、脐带来源的干细胞已在部分医院应用到脑性瘫痪患儿中，并取得了初步的疗效，其具体评定标准及长期疗效仍有待进一步随访、观察。