神经干细胞移植治疗小鼠机械性脑损伤的实验研究

目的探讨神经干细胞移植后的体内存活、增殖与分化,及其对小鼠机械性脑损伤的治疗作用。方法运用牙科钻制作小鼠运动区皮质机械性损伤模型。48只清洁级昆明小鼠,雌雄不拘,体质量为18～20g,按体质量编号随机分为4组:神经干细胞移植组(损伤后原位移植经鉴定确认的原代培养的小鼠神经干细胞)、3T3移植组(损伤后原位移植3T3细胞)、单纯损伤组(损伤后不行神经干细胞移植)和空白对照组(仅施行麻醉),每组12只小鼠。于伤后第3天进行行为学检测;第10、30天行损伤区脑组织nestin及NF200免疫荧光染色,观察神经干细胞生长、分化情况。结果损伤后,获得原代培养的神经干细胞在移植早期贴附于损伤区域且向周边组织呈浸润生长;移植后期Hoechst33342及NF200染色显示损伤区附近可见分化形成的神经元。单纯损伤组小鼠出现偏瘫症状;而神经干细胞移植组小鼠植入神经干细胞后则症状减轻,运动功能明显改善,与其他各组相比差异有显著性意义(P<0.001)。结论神经干细胞移植能够改善小鼠机械性脑损伤后的神经功能状态。     

外源性干细胞移植治疗缺血性脑损伤

全世界脑缺血性卒中患者大约有上千万例,每年都有近百万例新增患者.约半数患者都会遗留不同程度的神经功能缺失.干细胞移植为脑缺血性卒中患者提供了新的治疗方向.神经干细胞移植治疗脑缺血理想目标包括:(1)缺血区受损神经元能被问型神经元替代;(2)诱导局部血管再生使移植神经干细胞长期存活;(3)移植神经元能与周围细胞建立突触联系;f4)胶质细胞辅助移植神经元形成髓鞘.然而以上任何一点的完全实施都是极其困难的.     

神经干细胞治疗缺血性脑损伤研究进展

缺血性脑血管病是严重威胁人类健康的常见病和多发病,其病理过程涉及复杂的时间和空间级联反应,虽然对其机制的了解越来越深入,但有效的治疗手段仍然缺乏.对于大多数脑缺血患者,神经元死亡不可避免,是治疗难以取得满意效果的主要原因.近年来,已经从胚胎及成年的脑组织分离出具有自我更新、能够分化成神经元和胶质细胞的神经干细胞(neural stem cells, NSCs).因为NSCs能促进神经元再生及损伤脑组织恢复,许多学者开始研究NSCs对脑缺血的治疗作用,已经取得了重大进展.NSCs治疗缺血性脑损伤主要有两个途径:NSCs移植和自体NSCs原位激活.     

神经干细胞与缺血性脑损伤

介绍神经干细胞及其在缺血性脑损伤中的作用     

高压氧联合骨髓间充质干细胞移植修复缺氧缺血性脑损伤

背景：单纯骨髓间充质干细胞移植对脑梗死组织的修复作用并不理想,需要结合药物及生物工程材料等手段进行综合治疗。 目的：验证高压氧结合骨髓间充质干细胞移植修复大鼠缺氧缺血性脑损伤的效果。 方法：体外培养大鼠骨髓间充质干细胞。应用线栓法建立大脑中动脉阻塞大鼠模型,按随机区组法分为3组,即对照组、骨髓间充质干细胞移植组及高压氧+骨髓间充质干细胞移植组。静脉移植后24 h,3 d及伤后1,2 周行Longa行为学评分,检测神经功能的损伤情况。移植2周后,应用RT-PCR法测定生长相关蛋白43 mRNA的表达,并以BrdU免疫组化和苏木精-伊红染色行梗死处组织学检查以证实恢复程度。 结果与结论：移植后1周,高压氧+骨髓间充质干细胞移植组大鼠神经功能障碍评分低于骨髓间充质干细胞移植组,骨髓间充质干细胞移植组低于对照组(P < 0.05)。2周后脑梗死周围组织生长相关蛋白43 mRNA的表达高压氧+骨髓间充质干细胞移植组高于骨髓间充质干细胞移植组,骨髓间充质干细胞移植组高于对照组(P < 0.05)。BrdU免疫组化和苏木精-伊红切片中的神经元数量高压氧+骨髓间充质干细胞移植组多于骨髓间充质干细胞移植组,骨髓间充质干细胞移植组多于对照组(P < 0.05)。提示高压氧联合骨髓间充质干细胞静脉移植治疗大鼠缺氧缺血性脑损伤可明显改善大鼠的神经功能,效果优于单纯骨髓间充质干细胞移植。     

骨髓基质干细胞治疗缺血性脑损伤的实验研究

目的探讨脑缺血后,外源性植入的骨髓基质干细胞(bone marrow stromal cells,BMSCs)在脑内的迁移和分化,及脑缺血后自体神经干细胞(neural stromal cells,NSCs)的动员情况。方法体外培养、扩增BMSCs 后,用绿色荧光染料PKH67标记BMSCs,通过静脉和脑内局部移植等途径将BMSCs植入大脑中动脉闭塞的SD大鼠脑内,10d后取材,免疫荧光检测BMSCs的分化、迁移情况;并比较假手术和脑缺血时脑内自体NSCs的数量。结果 (1)静脉移植组两侧脑半球中PKH67 细胞数分别是46．4±2．92和21．8±1．13／100mm2(P<0．05);(2)移植的BMSCs中40．31％表达神经元特异性标志;(3)在第8天时,脑缺血半球和非脑缺血半球Nestin 细胞数分别为 19．5±10．08和7±1．41,差异显著(P<0．05)。结论脑缺血可激活脑内自身的NSCs,使它们迁移至脑缺血半球并促进其修复;外源性BMSCs通过局部或静脉移植后可迁移到缺血脑组织附近,并且部分分化为神经元样细胞,表达神经元的特异性标志。     

神经干细胞移植在脑损伤治疗中的应用

背景：成年鼠脑纹状体中已分离出具有分化成神经元及各种胶质细胞潜能的神经干细胞后,医学工作者试图从中寻找新思路用于脑损伤的治疗。目的：总结神经干细胞移植对治疗脑损伤中的作用及其前景。方法：电子检索EMbase(1980-01/2011-04),MEDLINE(1966-01/2011-04),中国生物医学文献数据库(CBM,1978/2011-04)和中国期刊全文数据库(CNKI),筛查相关文章的参考文献。中文检索词“神经干细胞,脑损伤,脑卒中,脑梗死”,英文检索词“neural stem cells, brain injury, stroke, cerebral infarction”,最终纳入符合标准的文献12篇。结果与结论：动物研究结果显示神经干细胞移植可以改善神经功能并且明显降低脑组织炎症反应,沉默NgR基因、GDNF基因修饰和联合移植BDNF可更大程度上恢复神经功能。临床试验发现神经干细胞移植能在一定程度上改善脑卒中患者的后遗症。提示神经干细胞移植在动物实验中获取明显效果,可进一步应用于临床并研究其疗效。     

神经干细胞移植治疗缺血性脑卒中研究进展

<正>脑血管疾病是全球面临的首要健康问题,缺血性脑卒(ischemic stroke)中发病率、致残率和死亡率一直居高不下。缺血性脑卒的核心病变是致血管内部空间变窄时甚至封闭血管,缺氧缺糖(oxygen-glucose deprivation,OGD)引发炎性损伤、组织坏死和细胞凋亡的发生~([1])。溶栓/切除血栓的临床治疗策略由于治疗时间窗狭窄难以达到满意的治疗效果,因此开发缺血性脑卒中精准治疗新策略势在必行。近年来研究发现神经干细胞(neural stem cells,NSCs)及骨髓间充质     

缺血性脑损伤的干细胞治疗研究

全球每年约有9％的死亡病例由脑卒中引起,而缺血性脑卒中的比例占80％,其发病率仅次于缺血性心脏疾病。因此对缺血性脑损伤进行有效防治具有重要的医学和社会意义。现经临床证实唯一能缩小梗死体积、减轻神经损伤的治疗药物是重组组织型纤溶酶原激活物（recombinant tissue plaminogen activator,t-PA）,且其有效治疗时间窗只有3h,     

骨髓间质干细胞移植治疗缺血性脑损伤的研究进展

脑血管疾病是目前世界三大主要致死疾病之一.而且由于成年哺乳动物的神经元缺乏再生能力,使其在损伤后很难恢复,这是临床上缺血性脑损伤难以取得满意疗效的主要原因.骨髓间质干细胞(mesenchymal stem cells,MSCs)是多能细胞,在一定诱导条件下可分化为多种造血外组织,特别是中胚层和神经外胚层发育来源的组织细胞,如成骨细胞、成软骨细胞、脂肪细胞、成肌细胞、以及神经细胞、神经胶质细胞等.另外它还具有易于获得,可从患者本人取材;体外培养可快速增殖;免疫原性阴性;移植后能与受体脑细胞结合并长期存活;以及能稳定转染并表达外源性基因产物等特点,所以是治疗缺血性脑损伤理想的移植细胞.     

神经干细胞移植对颅脑外伤神经组织的替代和修复作用

目的 神经干细胞具有自我更新及多向分化潜能的特性。本文将人类神经干细胞移植到大鼠颅脑损伤的模型中，观察是否有治疗效果。方法 人类神经干细胞取自3－4月胎儿海马，体重为300－370g的大鼠制成脑损伤模型，伤后24小时将神经干细胞用立体定向注射法注入双侧顶叶皮层，伤后1周动物运动功能评分后，处死取脑，行病理及免疫组化染色。结果 伤后1周接受干细胞移植的治疗组与损伤组相比呈现出明显的运动功能改善，一部分移植顶叶的干细胞呈Tubulin（＋）、GFAP（＋），表明它们分化成为神经元或胶质细胞。另外我们观察到治疗组伤区皮层的正常神经元增多，且坏死及凋亡的神经元减少。结论 社会干细胞是细胞移植治疗颅脑损伤的一种良好的细胞来源。     

Neural stem cells reduce brain injury after unilateral carotid ligation

Neonatal stroke presents with seizures and results in neurologic morbidity, including epilepsy, hemiparesis, and cognitive deficits. Stem cell-based therapy offers a possible therapeutic strategy for neonatal stroke. We developed an immature mouse model of stroke with acute seizures and ischemic brain injury. Postnatal day 12 CD1 mice received right-sided carotid ligation. Two or 7 days after ligation, mice received an intrastriatal injection of B5 embryonic stem cell-derived neural stem cells. Four weeks after ligation, hemispheric brain atrophy was measured. Pups receiving stem cells 2 days after ligation had less severe hemispheric brain atrophy compared with either noninjected or vehicle-injected ligated controls. Transplanted cells survived, but 3 out of 10 pups injected with stem cells developed local tumors. No difference in hemispheric brain atrophy was seen in mice injected with stem cells 7 days after ligation. Neural stem cells have the potential to ameliorate ischemic injury in the immature brain, although tumor development is a serious concern.     

干细胞移植救治颅脑创伤的再思考

近十多年来有关干细胞移植救治脑创伤的临床与基础研究方兴未艾。在取得一系列成果的同时,也带来了诸多问题。在临床应用之前,充分认识其内在规律和本质、完善移植条件、规范移植程序,以及客观合理评价治疗效果显得尤为重要。本文就该领域的研究予以简单回顾和讨论。     

立体定向移植神经干细胞治疗局灶性大鼠脑缺血损伤的研究

目的观察大鼠胚胎神经干细胞移植到局灶性脑缺血大鼠脑室内的迁移和分化。方法实验于2003年5月~2004年10月在哈尔滨医科大学附属二院动物实验中心完成。孕龄8~10d Wistar大鼠仔鼠神经干细胞体外扩增后用免疫组织化学的方法分别检测神经干细胞及其分化后代的特异性标志蛋白-巢蛋白、胶质纤维酸性蛋白和神经元特异性烯醇化酶的表达,健康Wistar大鼠大脑中动脉闭塞后40只随机分成A、B、C、D四组,A组为梗塞后24小时脑室内移植神经干细胞组,B组为梗塞后24小时梗塞中心移植神经干细胞组,C组为梗塞后4周脑室内移植神经干细胞组,D组为梗塞后4周梗塞中心移植神经干细胞组,各实验组均有仅移植生理盐水的空白对照。比较神经干细胞不同移植部位和不同移植时间神经干细胞存活、增殖和迁移的差异。结果实验大鼠40只均进入结果分析。从胎鼠中成功培养出悬浮生长的可表达巢蛋白的神经球,含血清条件下可分化为表达胶质纤维酸性蛋白的胶质细胞和神经元特异性烯醇化酶的神经元,移植后可见移植细胞存活至少8周以上,A组可见移植细胞大量存活,穿过室管膜向脑组织迁移,特别是向缺血周边区迁移;B组细胞主要聚集在梗塞中心移植区域,也有大量细胞存活,充填梗塞区,少量细胞可穿过胼胝体向健侧迁移;C组仅见少量Brdu阳性细胞存活,但细胞增殖分裂较A组不明显,且存活细胞主要仍在移植部位;D组存活移植细胞也较B组明显减少。结论大鼠胚胎神经干细胞移植到局灶性脑缺血大鼠脑室内可长期存活并广泛迁移,不同移植部位不影响细胞存活,脑室内移植细胞向脑缺血区域迁移,缺血后24小时移植较缺血后4周移植其迁移趋向性更强。     

间充质干细胞体内动员治疗缺血缺氧性脑损伤的研究进展

缺血缺氧性脑损伤是严重危害人类健康和生活质量的一类疾病.利用间充质干细胞(mesenchymal stem cells,MSCs)替代原来受损或功能缺陷的中枢神经组织治疗神经系统疾病目前已成为颇具潜力的治疗方法[1].MSCs具有获取方便、易于分离培养和扩增纯化、体外反复传代仍能保持多分化潜能的特点,且遗传背景稳定,其自体移植也克服了伦理道德和免疫排斥等问题[2].     

神经干细胞移植治疗脊髓损伤

目的:探讨神经干细胞移植对脊髓损伤大鼠后肢运动功能修复的影响。方法:SD大鼠36只,制成T10脊髓全横断损伤模型。于造模成功后1周采用局部微量注射法移植。随机分三组：A损伤对照组（n=12）仅打开椎管暴露脊髓;B移植对照组（n=12）：注射10μl DMEM/F12培养液;C细胞移植组（n=12）：移植1.0?06/ml的神经干细胞悬液10μl。移植后通过不同时间点BBB行为评分、病理组织学、免疫荧光技术评价大鼠大鼠脊髓功能修复情况及移植细胞在体内的存活、迁移、分化。 结果:在体外成功建立SD大鼠海马源性神经干细胞培养体系;B、C两组大鼠随着时间延长BBB评分均不同程度提高,从移植后2W起C组大鼠评分明显高于B组,两组比较差异有统计学意义（P<0.05）;神经干细胞移植后能够在体内继续存活、迁移并且分化为NF-200、GFAP表达阳性的神经元及星形胶质细胞。 结论:神经干细胞移植治疗脊髓损伤是一种有效的方法。     

Intravenous transplantation of bone marrow mesenchymal stem cells promotes neural regeneration after traumatic brain injury

To investigate the supplement of lost nerve cells in rats with traumatic brain injury by intravenous administration of allogenic bone marrow mesenchymal stem cells, this study established a Wistar rat model of traumatic brain injury by weight drop impact acceleration method and administered 3 × 106 rat bone marrow mesenchymal stem cells via the lateral tail vein. At 14 days after cell transplantation, bone marrow mesenchymal stem cells differentiated into neurons and astrocytes in injured rat cerebral cortex and rat neurological function was improved significantly. These findings suggest that intravenously administered bone marrow mesenchymal stem cells can promote nerve cell regeneration in injured cerebral cortex, which supplement the lost nerve cells.     

Magnet-targeted delivery of bone marrow-derived mesenchymal stem cells improves therapeutic efficacy following hypoxic-ischemic brain injury

hypoxicischemic brain injury;however,the therapeutic efficacy of bone marrow-derived mesenchymal stem cells largely depends on the number of cells that are successfully transferred to the target.Magnet-targeted drug delivery systems can use a specific magnetic field to attract the drug to the target site,increasing the drug concentration.In this study,we found that the double-labeling using superparamagnetic iron oxide nanoparticle and poly-L-lysine(SPIO-PLL)of bone marrow-derived mesenchymal stem cells had no effect on cell survival but decreased cell proliferation 48 hours after labeling.Rat models of hypoxic-ischemic brain injury were established by ligating the left common carotid artery.One day after modeling,intraventricular and caudal vein injections of 1×105 SPIO-PLL-labeled bone marrow-derived mesenchymal stem cells were performed.Twenty-four hours after the intraventricular injection,magnets were fixed to the left side of the rats’heads for 2 hours.Intravoxel incoherent motion magnetic resonance imaging revealed that the perfusion fraction and the diffusion coefficient of rat brain tissue were significantly increased in rats treated with SPIO-PLL-labeled cells through intraventricular injection combined with magnetic guidance,compared with those treated with SPIO-PLL-labeled cells through intraventricular or tail vein injections without magnetic guidance.Hematoxylin-eosin and terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)staining revealed that in rats treated with SPIO-PLL-labeled cells through intraventricular injection under magnetic guidance,cerebral edema was alleviated,and apoptosis was decreased.These findings suggest that targeted magnetic guidance can be used to improve the therapeutic efficacy of bone marrow-derived mesenchymal stem cell transplantation for hypoxic-ischemic brain injury.This study was approved by the Animal Care and Use Committee of The Second Hospital of Dalian Medical University,China(approval No.2016-060)on March 2,2016.     

Neural stem cells may be uniquely suited for combined gene therapy and cell replacement: Evidence from engraftment of Neurotrophin-3-expressing stem cells in hypoxic-ischemic brain injury

Previously, we reported that, when clonal neural stem cells (NSCs) were transplanted into brains of postnatal mice subjected to unilateral hypoxic-ischemic (HI) injury (optimally 3-7 days following infarction), donor-derived cells homed preferentially (from even distant locations) to and integrated extensively within the large ischemic areas that spanned the hemisphere. A subpopulation of NSCs and host cells, particularly in the penumbra, "shifted" their differentiation towards neurons and oligodendrocytes, the cell types typically damaged following asphyxia and least likely to regenerate spontaneously and in sufficient quantity in the "post-developmental" CNS. That no neurons and few oligodendrocytes were generated from the NSCs in intact postnatal cortex suggested that novel signals are transiently elaborated following HI to which NSCs might respond. The proportion of "replacement" neurons was approximately 5%. Neurotrophin-3 (NT-3) is known to play a role in inducing neuronal differentiation during development and perhaps following injury. We demonstrated that NSCs express functional TrkC receptors. Furthermore, the donor cells continued to express a foreign reporter transgene robustly within the damaged brain. Therefore, it appeared feasible that neuronal differentiation of exogenous NSCs (as well as endogenous progenitors) might be enhanced if donor NSCs were engineered prior to transplantation to (over)express a bioactive gene such as NT-3. A subclone of NSCs transduced with a retrovirus encoding NT-3 (yielding >90% neurons in vitro) was implanted into unilaterally asphyxiated postnatal day 7 mouse brain (emulating one of the common causes of cerebral palsy). The subclone expressed NT-3 efficiently in vivo. The proportion of NSC-derived neurons increased to approximately 20% in the infarction cavity and >80% in the penumbra. The neurons variously differentiated further into cholinergic, GABAergic, or glutamatergic subtypes, appropriate to the cortex. Donor-derived glia were rare, and astroglial scarring was blunted. NT-3 likely functioned not only on donor cells in an autocrine/paracrine fashion but also on host cells to enhance neuronal differentiation of both. Taken together, these observations suggest (1) the feasibility of taking a fundamental biological response to injury and augmenting it for repair purposes and (2) the potential use of migratory NSCs in some degenerative conditions for simultaneous combined gene therapy and cell replacement during the same procedure in the same recipient using the same cell (a unique property of cells with stem-like attributes).     

Human insulin-like growth factor 1-transfected umbilical cord blood neural stem cell transplantation improves hypoxic-ischemic brain injury

Human insulin-like growth factor 1-transfected umbilical cord blood neural stem cells were transplanted into a hypoxic-ischemic neonatal rat model via the tail vein. BrdU-positive cells at day 7 post-transplantation, as well as nestin- and neuron specific enolase-positive cells at day 14 were increased compared with those of the single neural stem cell transplantation group. In addition, the proportion of neuronal differentiation was enhanced. The genetically modified cell-transplanted rats exhibited enhanced performance in correctly crossing a Y-maze and climbing an angled slope compared with those of the single neural stem cell transplantation group. These results showed that human insulin-like growth factor 1-transfected neural stem cell transplantation promotes the recovery of the learning, memory and motor functions in hypoxic-ischemic rats.