神经干细胞和骨髓间充质干细胞移植治疗脊髓损伤效果与机制对比

目的比较神经干细胞和骨髓间充质干细胞移植治疗脊髓损伤的机制及实验效果。方法选择40只Wistar成年大鼠做脊髓半横切模型,随机分为神经干细胞注射组,骨髓间充质干细胞注射组,磷酸盐缓冲液注射组和假手术组,每组10只。对比4组大鼠移植后的运动功能和脊髓损伤的修复情况。结果神经干细胞注射组各个时间点的BBB评分明显高于骨髓间充质干细胞注射组,且2组BBB评分明显高于磷酸盐缓冲液注射组,差异具有统计学意义(P0.05);神经干细胞和骨髓间充质干细胞移植后的第8周,MRI显示空洞明显缩小,信号强度正常,能看到完整的脊髓,脊髓切片中能看到被标记的NSCs及BMSCs。结论脊髓损伤大鼠通过静脉注射NSCs和BMSCs均能改善运动功能,但NSCs治疗效果更为明显,应将两种方法结合起来,进一步提高治疗效果。     

骨髓间充质干细胞移植脊髓全横断模型大鼠运动和体感诱发电位的评价

背景：临床常用皮质运动诱发电位和皮质体感诱发电位来分别评价脊髓损伤后运动传导路和感觉传导路的损伤或修复情况。目的：以脊髓诱导电位监测骨髓间充质干细胞移植后急性脊髓完全性损伤大鼠下肢神经功能的变化。方法：选取健康Wistar大鼠50只,分成5组,即生理盐水组、骨髓间充质干细胞移植组、脑源性神经营养因子修饰组、神经营养素3+骨髓间充质干细胞移植组和假手术组。除假手术组外,其余各组均制作Allen’s脊髓完全性损伤动物模型,造模后各组均行相应治疗。治疗后4,8和12周行大鼠后肢运动功能评分,并于造模后24 h,3,7,14 d行运动和体感诱发电位检测。结果与结论：运动诱发电位检测结果提示,各治疗组的运动功能均有不同程度的恢复,与生理盐水组间差异均有显著性意义(P < 0.05),大鼠后肢BBB评分也证实了各治疗组后肢运动功能明显优于生理盐水组(P < 0.05)。提示经脑源性神经营养因子修饰的骨髓间充质干细胞可移植到脊髓损伤处,可改善大鼠的后肢运动,神经营养素3蛋白有可能提高骨髓间充质干细胞在体内的生存率,促进受损脊髓的轴突再生。     

神经干细胞与许旺细胞共移植于大鼠损伤脊髓

目的 观察神经干细胞与许旺细胞共移植于大鼠半横断脊髓损伤处神经干细胞的迁移、存活、分化及对损伤脊髓的修复作用.方法 绿色荧光蛋白(GFP)标记脊髓神经下细胞后与许旺细胞共移植于大鼠半横断脊髓损伤处,免疫荧光染色和电镜技术分别观察神经下细胞的迁移、存活、分化及新生的髓鞘.皮层运动诱发电位(CMEPs)及BBB评分分别检测大鼠运动功能的恢复.结果 在神经干细胞与许旺细胞共移植组,损伤脊髓的头端、尾端及对侧町见明显的GFP阳性细胞及GaLC/GFP、GFAP/GFP、NSE/GFP、SYN/GFP舣阳性细胞,电镜下新生的髓鞘最多,CMEPs恢复百分率和振幅明显高于其他两组,但BBB评分与神经干细胞单移植组差异无统计学意义.结论 神经干细胞和许旺细胞体内共移植可促进神经干细胞的辽移、存活、分化及脊髓运动功能的恢复.     

补体系统抑制剂联合骨髓间充质干细胞移植用于修复脊髓损伤大鼠的研究

目的研究一种补体系统抑制剂与骨髓间充质干细胞联合移植用以治疗脊髓损伤大鼠的方法。方法体外分离、培养、标记大鼠骨髓间充质干细胞,并加入补体系统抑制剂制成二者的混合悬液;在立体定位仪下分别于损伤区中心及远、近端各1 mm处三点注射于重物打击致脊髓全瘫Allen模型大鼠体内,观察术后体重变化,BBB评分,BMSC存活免疫组织化学观察及计数,并对MPO活性进行测定。结果补体系统抑制剂与骨髓间充质干细胞联合移植的方法体重恢复最快,BBB评分最高,BMSC存活免疫组织化学观察及计数结果、MPO活性等指标均优于骨髓间充质干细胞单独移植组,且都优于空白对照组。结论补体系统抑制剂与骨髓间充质干细胞联合移植用以治疗脊髓损伤的方法具有一定效果,值得深入研究。     

骨髓间充质干细胞尾静脉移植脊髓损伤大鼠脑源性神经营养因子及神经生长因子的表达

背景：骨髓间充质干细胞移植对脊髓损伤有治疗作用,但其机制尚不完全清楚。 目的：应用免疫组织化学方法观察骨髓间充质干细胞静脉移植损伤脊髓局部脑源性神经营养因子及神经生长因子的表达,分析骨髓间充质干细胞移植治疗大鼠脊髓损伤的作用途径。 方法：运用改良Allen法制备T10脊髓外伤性截瘫大鼠模型,假手术组6只,脊髓损伤组24只随机分为对照组和骨髓间充质干细胞移植组。骨髓间充质干细胞移植组、假手术组接受骨髓间充质干细胞单细胞悬液1 mL(1×106 cells)自大鼠尾静脉缓慢注射移植,对照组静脉注射PBS 1 mL。 结果与结论：脊髓损伤后损伤局部的脑源性神经营养因子、神经生长因子表达增加,骨髓间充质干细胞静脉注射移植后能促进脊髓损伤局部脑源性神经营养因子、神经生长因子更进一步的表达,这可能是促进神经结构及神经功能恢复的因素之一。     

大鼠骨髓间充质干细胞静脉移植对脊髓损伤的修复作用

目的初步探讨骨髓间充质干细胞（BMSCs）静脉移植对脊髓损伤后神经功能恢复和神经修复的影响。方法体外培养BMSCs，改良Allen法制备大鼠脊髓损伤模型，经尾静脉移植Brdu标记的BMSCs，损伤后24h、移植后1、3、5周评价实验动物的神经功能状况，并检测BMSCs在体内迁移、存活以及分化情况，电子显微镜观察组织形态学变化。结果移植的BMSCs在宿主损伤脊髓中聚集并存活，3～5周后有部分移植细胞表达神经元特异性烯醇化酶（NSE）、神经丝蛋白（NF）、微管相关蛋白（MAP2）；BMSCs静脉移植组大鼠运动功能改善，BBB评分高于对照组（P〈0．05）；5周后组织学观察，与对照组相比移植组损伤区脊髓结构较完整。结论BMSCs经静脉移植后可向脊髓损伤处聚集并存活分化，促进神经修复及神经功能的恢复。     

嗅鞘细胞移植联合应用督脉电针对大鼠脊髓损伤后脊髓诱发电位的影响※

背景：对于嗅鞘细胞移植治疗脊髓损伤的疗效目前尚无共识,或许单一细胞移植可能并不是修复脊髓损伤的最佳选择。如何选择适当的干预手段予以联合应用,并使之实现从实验室走向临床应用是细胞移植策略中的重点问题。 目的：探讨大鼠嗅鞘细胞移植与督脉电针联合应用修复大鼠脊髓损伤的可行性。 设计、时间及地点：随机对照动物实验,于2007-08/2008-08在清华大学二附院脑神经病研究所实验室完成。 材料：成年雄性Wistar大鼠70只,取10只用于制备嗅鞘细胞,剩余60只随机分为4组：模型对照组、嗅鞘细胞移植组、督脉电针组、联合组,15只/组。 方法：各组大鼠均建立脊髓全横断模型。造模后暴露脊髓,嗅鞘细胞移植组、联合组向填入脊髓横断处的明胶海绵内缓慢注射嗅鞘细胞悬液10 μL;模型对照组、督脉电针组同法注射等量DMEM-F12培养液。从造模成功后第2天开始,督脉电针组、联合组动物接受1次/d的督脉电针治疗,选大椎穴(DU14)、命门穴(DU4)进行针刺,针刺深度5 mm,大椎穴接正极,命门穴接负极,电针15 min,电针频率20 Hz,持续脉冲电流12~15 mV,连续7 d为1个疗程,疗程间隔2 d。 主要观察指标：造模后BBB运动功能评分的变化,脊髓诱发电位检测结果。 结果：各组动物均成活10周。与模型对照组比较,造模后4~10周嗅鞘细胞移植组、督脉电针组、联合组BBB运动功能评分均明显升高(P < 0.05),且联合组升高幅度明显高于嗅鞘细胞移植组、督脉电针组(P < 0.05)。与模型对照组比较,造模后4~10周嗅鞘细胞移植组、督脉电针组、联合组波幅电压明显升高(P < 0.05或P < 0.01),反应潜伏期均明显降低(P < 0.05或P < 0.01),且联合组差异变化尤为显著。嗅鞘细胞移植组与督脉电针组各指标之间比较无明显差异(P > 0.05)。 结论：嗅鞘细胞移植和督脉电针联合应用可促进脊髓损伤大鼠神经突触的再生,改善其肢体运动功能。     

膀胱护理对脊髓损伤致神经源性膀胱患者的影响

随着社会的发展、人口的增多、交通事故的增多,脊髓损伤（SCI）的患者日益增多.脊髓损伤[1]患者由于损伤的部位不同,可出现不同程度的肢体运动感觉障碍及排尿障碍,严重影响患者的生活质量[1].神经源性膀胱是脊髓损伤患者的并发症之一,易导致尿潴留、肾积水、感染、慢性肾衰竭、死亡等,给家庭及社会带来了沉重的负担及压力.我科及康复科应用膀胱护理治疗50例脊髓损伤致神经源性膀胱的患者,取得良好疗效,现报告如下.     

大鼠脊髓损伤模型建立及神经源性肠功能障碍的初步评价

目的 探讨建立大鼠脊髓损伤模型的方法,并初步评价神经源性肠功能障碍的发生、发展及变化规律。方法 56只大鼠随机分为脊髓损伤组（n=40）、假手术组（n=8）和正常组（n=8）,其中脊髓损伤组再根据损伤后时间分为伤后1 d组、3 d组、7 d组、14 d组、28 d组等5个亚组,每亚组8只。假手术组和正常组合称为非脊髓损伤组。采用自制改良Allen脊髓撞击器造成脊髓不完全性损伤,假手术组只暴露硬膜囊。采用肠道推进试验、粪便粒数及粪便含水率测定来评价脊髓损伤后肠功能情况。结果 与非脊髓损伤组相比,脊髓损伤组结肠推进率明显降低（P<0.05）;而且,脊髓损伤后1 d组结肠推进率降低最明显（P<0.05）。与非脊髓损伤组相比,脊髓损伤组粪便粒数明显增多（P<0.05）;而且,脊髓损伤后1 d组粪便粒数最多（P<0.05）。与非脊髓损伤组相比,脊髓损伤组粪便含水率明显降低（P<0.05）;而且,脊髓损伤后1 d组粪便含水率降低最明显（P<0.05）。结论 采用自制改良Allen脊髓撞击器能成功复制大鼠不完全性脊髓损伤模型;大鼠脊髓损伤后出现神经源性肠功能障碍,损伤后急性期（伤后1 d）尤为显著,随后逐渐改善。     

大鼠脊髓源性神经干细胞的培养分化及其特异性研究

目的 研究大鼠脊髓源性神经干细胞培养和分化的特异性。方法 从孕17d的SD大鼠胚胎脊髓中分离，培养神经干细胞并用血清诱导其分化，通过免疫荧光化学方法研究其特性。结果 在血清的诱导下，脊髓源性神经干细胞大多数分化成GFAP阳性的星形胶质细胞，少数分化为tubulin-β阳性的神经细胞；与脑源性神经干细胞分化的神经细胞相比较，其分化出的神经细胞的突起长度明显延长。结论 脊髓源性神经干细胞在体外具有多向分化潜能，但与脑源性神经干细胞有明显差别。     

大鼠骨髓间质干细胞静脉移植在脊髓损伤中的定向迁移

目的观察大鼠骨髓间质干细胞(ratbonemarrowstromalcells,rMSCs)静脉移植在体内的定向迁移。方法分离培养rMSCs,流式细胞术检测其表面标志,运用改良Allen法制备大鼠T10脊髓损伤模型,随机分为假手术组、对照组、rMSCs静脉移植组。假手术组、rMSCs静脉移植组同时于大鼠损伤后72小时经尾静脉移植溴脱氧尿苷(BrdU)标记MSCs。免疫组化技术检测rMSCs在体内迁移、存活以及分化情况。结果rMSCs体外分离培养扩增5代,细胞数可达1～2×1011个,具有多态性和贴壁生长特性,流式细胞术检测CD34、CD45阴性,CD29、CD90表达阳性。移植的rMSCs在宿主损伤脊髓中聚集并存活,3～5W后即有部分移植细胞表达神经元特异性烯醇化酶(neuronspecificenolase,NSE)、神经丝蛋白(neurofilament,NF)、微管相关蛋白(microtubuleassociatedprotein2,MAP2)。结论rMSCs体外扩增迅速,具有干细胞特性,经静脉移植在宿主体内可向损伤区脊髓聚集存活分化。     

Transplantation of human bone marrow stromal cell‐derived Schwann cells reduces cystic cavity and promotes functional recovery after contusion injury of adult rat spinal cord

The aim of this study was to evaluate whether transplantation of human bone marrow stromal cell‐derived Schwann cells (hBMSC‐SC) promotes functional recovery after contusive spinal cord injury of adult rats. Human bone marrow stromal cells (hBMSC) were cultured from bone marrow of adult human patients and induced into Schwann cells (hBMSC‐SC) in vitro. Schwann cell phenotype was confirmed by immunocytochemistry. Growth factors secreted from hBMSC‐SC were detected using cytokine antibody array. Immunosppressed rats were laminectomized and their spinal cords were contused using NYU impactor (10 g, 25 mm). Nine days after injury, a mixture of Matrigel and hBMSC‐SC (hBMSC‐SC group) was injected into the lesioned site. Five weeks after transplantation, cresyl‐violet staining revealed that the area of cystic cavity was smaller in the hBMSC‐SC group than that in the control group. Immunohistochemstry revealed that the number of anti‐growth‐associated protein‐43‐positive nerve fibers was significantly larger in the hBMSC‐SC group than that in the control group. At the same time, the number of tyrosine hydroxylase‐ or serotonin‐positive fibers was significantly larger at the lesion epicenter and caudal level in the hBMSC‐SC group than that in the control group. In electron microscopy, formation of peripheral‐type myelin was recognized near the lesion epicenter in the hBMSC‐SC group. Hind limb function recovered significantly in the hBMSC‐SC group compared with the control group. In conclusion, the functions of hBMSC‐SC are comparable to original Schwann cells in rat spinal cord injury models, and are thus potentially useful treatments for patients with spinal cord injury.     

BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury

Bone marrow stromal cells (MSCs) constitute a heterogeneous cell layer in the bone marrow, supporting the growth and differentiation of hematopoietic stem cells. Recently, it has been reported that MSCs harbor pluripotent stem cells capable of neural differentiation and that simple treatment of MSCs with chemical inducing agents leads to their rapid transdifferentiation into neural cells. We examined whether native or neurally induced MSCs would reconstitute an axonal growth-promoting milieu after cervical spinal cord injury (SCI), and whether such cells could act as vehicles of growth factor gene delivery to further augment axonal growth. One month after grafting to cystic sites of SCI, native MSCs supported modest growth of host sensory and motor axons. Cells "neurally" induced in vitro did not sustain a neural phenotype in vivo and supported host axonal growth to a degree equal to native MSCs. Transduction of MSCs to overexpress brain-derived neurotrophic factor (BDNF) resulted in a significant increase in the extent and diversity of host axonal growth, enhancing the growth of host serotonergic, coerulospinal, and dorsal column sensory axons. Measurement of neurotrophin production from implanted cells in the lesion site revealed that the grafts naturally contain nerve growth factor (NGF) and neurotrophin-3 (NT-3), and that transduction with BDNF markedly raises levels of BDNF production. Despite the extensive nature of host axonal penetration into the lesion site, functional recovery was not observed on a tape removal or rope-walking task. Thus, MSCs can support host axonal growth after spinal cord injury and are suitable cell types for ex vivo gene delivery. Combination therapy with other experimental approaches will likely be required to achieve axonal growth beyond the lesion site and functional recovery.     

Bone marrow stromal cells infused into the cerebrospinal fluid promote functional recovery of the injured rat spinal cord with reduced cavity formation

The effects of bone marrow stromal cells (BMSCs) on the repair of injured spinal cord and on the behavioral improvement were studied in the rat. The spinal cord was injured by contusion using a weight-drop at the level of T8-9, and the BMSCs from the bone marrow of the same strain were infused into the cerebrospinal fluid (CSF) through the 4th ventricle. BMSCs were conveyed through the CSF to the spinal cord, where most BMSCs attached to the spinal surface although a few invaded the lesion. The BBB score was higher, and the cavity volume was smaller in the rats with transplantation than in the control rats. Transplanted cells gradually decreased in number and disappeared from the spinal cord 3 weeks after injection. The medium supplemented by CSF (250 microl in 3 ml medium) harvested from the rats in which BMSCs had been injected 2 days previously promoted the neurosphere cells to adhere to the culture dish and to spread into the periphery. These results suggest that BMSCs can exert effects by producing some trophic factors into the CSF or by contacting with host spinal tissues on the reduction of cavities and on the improvement of behavioral function in the rat. Considering that BMSCs can be used for autologous transplantation, and that the CSF infusion of transplants imposes a minimal burden on patients, the results of the present study are important and promising for the clinical use of BMSCs in spinal cord injury treatment.     

骨髓基质细胞源内皮细胞对大鼠局灶性脑损伤超微结构的影响

目的研究骨髓基质细胞源内皮细胞移植对大鼠局灶性脑损伤超微结构的影响,探讨骨髓基质细胞源内皮细胞移植修复大鼠脑损伤的机制。方法制备大鼠局灶性脑损伤动物模型,进行骨髓基质细胞源内皮细胞移植,通过透射电镜观察损伤局部超微结构的变化。结果脑损伤后微血管内皮细胞胞浆广泛空泡变,细胞器明显减少,并出现核固缩,血管壁破坏。骨髓基质细胞源内皮细胞移植后微循环得到改善。结论脑损伤后出现微循环障碍是产生继发性脑缺血、脑水肿的病理基础。骨髓基质细胞源内皮细胞移植促进了损伤区微循环的改善。     

骨髓基质细胞源内皮样细胞对微血管新生的影响

目的研究骨髓基质细胞(BMSCs)源内皮样细胞移植对大鼠局灶性脑损伤血管生成的影响,探讨BMSCs源内皮样细胞移植修复大鼠脑损伤的机制。方法制备大鼠局灶性脑损伤动物模型,进行BMSCs源内皮样细胞移植,通过ELISA、免疫组织化学、电镜等观察局部血管内皮细胞生长因子(VEGF)、内皮细胞及微循环的变化。结果 BMSCs源内皮样细胞移植后,局部VEGF水平升高、凝血因子FⅧ染色阳性细胞数增加,染色加深;局部微血管内膜较光滑,细胞核形态较规则,基底膜较完整,微血管管腔受压缓解。结论 BMSCs源内皮样细胞移植促进损伤区周围内皮细胞增殖和新生血管形成,损伤区局部微循环得以改善。     

Acellular spinal cord scaffold seeded with bone marrow stromal cells protects tissue and promotes functional recovery in spinal cord‐injured rats

Therapy using scaffolds seeded with stem cells plays an important role in repair of spinal cord injury (SCI), with the transplanted cells differentiating into nerve cells to replace the lost tissue while releasing neurotrophic factors that contribute to repair following SCI and enhance the function of the damaged nervous system. The present study investigated the ability to extend the survival time of bone marrow stromal cells (BMSCs) to restore the damaged spinal cord and improve functional recovery by grafting acellular spinal cord (ASC) scaffold seeded or not with BMSCs in a rat model of acute hemisected SCI. BBB scores revealed that treatment with BMSCs seeded into ASC scaffold led to an obvious improvement in motor function recovery compared with treatment with ASC scaffold alone or untreated controls. This improvement was evident at 2 and 8 weeks after surgery (P < 0.05). When BMSCs labeled with 5‐bromodeoxyuridine were implanted together with ASC scaffold into the injured sites, they differentiated into glial cells, and some BMSCs could be observed within the graft by immunofluorescent staining at 8 weeks after implantation. Evaluation of caspase‐3 activation suggested that the graft group was able to reduce apoptosis compared with SCI alone at 8 weeks after operation (P < 0.05). This study suggests that ASC scaffolds have the ability to enhance BMSC survival and improve differentiation and could also reduce native damaged nerve tissue apoptosis, thus protecting host tissue as well as improving functional recovery after implantation. © 2013 Wiley Periodicals, Inc.