人羊膜间充质干细胞移植对大鼠脊髓损伤神经功能恢复的影响

目的 观察人羊膜间充质干细胞( hAD - MSCs)移植对大鼠脊髓损伤神经功能恢复的影响.方法 建立大鼠脊髓全横断损伤模型,脊髓横断后立即以明胶海绵吸附10μl hAD- MSCs(约2×105个)或等量PBS液植入脊髓两断端之间.术后每周应用BBB评分评价大鼠后肢运动功能;采用免疫荧光染色观察hAD - MSCs在脊髓内的存活、分化情况;免疫组织化学染色观察受损脊髓远端组织NF - 200表达.结果 hAD - MSCs移植组神经功能明显恢复,BBB评分逐周增加,与对照组比较差异有统计学意义(P＜0.05).hAD - MSCs植入后2周在宿主脊髓中存在MAB1281染色阳性细胞,但不表达MAP -2和GFAP.hAD - MSCs移植后大鼠受损脊髓远端神经组织NF - 200表达明显强于对照组.结论 hAD - MSCs移植可促进大鼠脊髓损伤后的神经功能恢复,其机制可能与hAD - MSCs促进受损脊髓远端组织表达NF - 200有关.     

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。     

移植人脐带间充质干细胞修复大鼠脊髓损伤

背景：已知人脐带间充质干细胞对脊髓损伤存在着潜在的治疗价值,然而,当前对移植人脐带间充质干细胞治疗脊髓损伤及机制方面研究很少。 目的：观察人脐带间充质干细胞对脊髓损伤大鼠的治疗效果。 方法：40只Wistar大鼠建立脊髓损伤模型,38只造模成功后随机摸球法分为3组：空白对照组：只接受单纯损伤,不做任何移植;DMEM移植组：损伤后1周予以5 μL DMEM局部移植;细胞移植组：损伤后1周予以5 μL准备好的人脐带间充质干细胞局部移植(细胞数1×106)。移植后对实验动物通过BBB评分、体感诱发电位与运动诱发电位观察后肢功能恢复情况。分别于损伤后2,4,6,8,10周随机于细胞移植组抽取大鼠2只,免疫组织化学染色观察人脐带间充质干细胞存活、迁移、分化,通过胶质纤维酸性蛋白阳性细胞染色比较各组损伤局部胶质瘢痕形成面积。 结果与结论：BBB评分损伤后4周细胞移植组高于其他两组(P < 0.05),损伤后12周细胞移植组与其他两组相比SEP、MEP潜伏期缩短、波幅值增高(P < 0.05)。免疫组织化学染色示人脐带间充质干细胞可向神经元、星形胶质细胞和少突胶质细胞分化,分化的少突胶质细胞并包绕轴突形成髓鞘。细胞移植组损伤局部胶质瘢痕面积均小于其他两组(P < 0.05),空白对照组、DMEM移植组间差异无显著性(P > 0.05)。提示未经体外诱导的人脐带间充质干细胞可于损伤大鼠脊髓体内向神经元、星形胶质细胞、少突胶质细胞分化,减小胶质瘢痕,并促进脊髓损伤大鼠神经功能的恢复。     

骨髓间质干细胞移植对大鼠脊髓损伤神经功能恢复的影响

目的：观察成人骨髓间质干细胞（hBMSCs)移植对大鼠脊髓损伤神经功能恢复的影响.方法：Wistar大鼠90只，随机分为脊髓半切＋hBMSCs组、脊髓半切＋PBS组、单纯脊髓半切组和假手术组。脊髓半切＋hBMSCs组和PBS组又分别分为头侧注射、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，应用免疫组化和免疫荧光技术检测BrdU标记hBMSCs的胶质纤维酸性蛋白（GFAP）和神经元特异性核蛋白（NeuN)表达情况。结果：大鼠脊髓半切损害后，hBMSCs组动物较PBS组死亡率下降并有明显的神经功能恢复。移植的hBMSCs 在宿主脊髓中存活，从第7天开始即有NeuN和GFAP表达并向损伤部位及对侧迁移，第28天hBMSCs来源GFAP阳性细胞可见明显的树突生长。结论：hBMSCs可在宿主损伤脊髓中存活、向损伤部位迁移并向神经元和星形胶质细胞分化，并促进神经功能恢复，降低死亡率，成人骨髓间质干细胞作为一种独特的干细胞来源用于治疗脊髓损伤可能具有非常重要的价值。     

人脐血间充质干细胞移植促进大鼠脊髓损伤康复的研究

目的 观察人脐血间充质干细胞(MSCs)对大鼠脊髓损伤的治疗作用,为脊髓损伤的临床治疗提供新策略.方法 分离纯化人脐血MSCs,制备大鼠脊髓损伤模型,随机分为假手术组、模型组和MSCs移植组(BrdU标记),移植后7、14、21d,采用BBB评分评估大鼠行为学变化,免疫荧光法检测MSCs的迁移、存活情况,免疫组化法检测炎症冈子HMGB1、NF-kB及凋亡蛋白Caspase-3在脊髓损伤部位的表达变化.结果 移植后21d,MSCs移植组大鼠肢体功能恢复明显,与模型组比较差别有统计学意义(P<0.05).MSCs移植组大鼠脊髓损伤区及周边可见BrdU阳性细胞.相同时间点,MSCs移植组HMGB1、NF-kB和Caspase-3的阳性表达率远低于模型组,差别有统计学意义(P<0.05).结论 人脐血MSCs移植可显著促进大鼠脊髓损伤的康复,这可能为脊髓损伤的临床治疗提供一种新的途径.     

大鼠骨髓间充质干细胞源性神经元促进脊髓横断性损伤的神经功能恢复

目的探讨干细胞治疗外伤性脊髓损伤的策略。方法体外分离纯化成年SD大鼠骨髓MSCs,并在体外培养过程中加入麝香多肽(Musk-1)将其诱导分化为神经前体细胞,再定向将神经前体细胞植入经显微外科手术建立的大鼠横断性脊髓损伤病灶中。结果与对照组大鼠相比,植入的rMSCs源性神经元可明显促进脊髓损伤后的神经功能恢复(P<0．05;有效观察期90 d)。组织学和免疫细胞组化分析进一步证实了植入rMSCs源性神经元在移植区域大量成活,并向损伤区域四周的邻近组织迁移约6 mm。荧光金逆行追踪分析显示在大鼠脊髓头侧、中脑红核和大脑感觉运动皮层等区域均可检测到荧光金标记阳性的运动神经元,推测脊髓损伤侧的皮层脊髓束发生了再生并穿越横断性病灶达到了脊髓尾侧。结论作为干细胞替代治疗的新策略,rMSCs源性神经元可在横断性脊髓损伤病灶中成活、迁移、整合,以及具备修补脊髓功能的潜在可能性。     

间充质干细胞移植修复脊髓损伤的研究现状

骨髓间充质干细胞在体外没有外界因素作用下连续传50代仍可保持原来的特性,亦可在一些条件的诱导下分化成神经细胞。实验研究表明,间充质干细胞移植能促进脊髓损伤的神经结构修复及神经功能恢复,其机制可能与替代作用、营养作用、诱导作用、桥接作用等有关。携带外源功能基因的骨髓间充质干细胞移植到体内存活、迁徙、分化并基因表达脑源性生长因子、神经生长因子等细胞因子,明显促进脊髓损伤的恢复,成为了新的研究热点。随着对间充质干细胞的生物学特性进一步深入研究,一些未解决的问题将会逐步得到解决,为脊髓损伤和其他神经系统疾病的患者带来新的希望。     

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。     

常用脊髓损伤模型与骨髓间充质干细胞移植治疗脊髓损伤

脊髓损伤的病理生理机制非常复杂,建立适宜的脊髓损伤模型,对模型进行骨髓间充质干细胞移植治疗,并分析其治疗脊髓损伤的机制,是进行临床脊髓损伤治疗的前提。目前常用的脊髓损伤模型包括挫伤型模型、牵张损伤模型、压迫损伤模型、切割或吸除型模型、缺血损伤模型等。常用的骨髓间充质干细胞移植方法有细胞悬液立体定位注射法、腰穿细胞悬液注射法、静脉内细胞悬液输入法等。骨髓间充质干细胞治疗脊髓损伤的机制可能有以下几方面：①骨髓间充质干细胞能向损伤处迁移,并向神经细胞表型分化。②发挥桥梁介导作用。③骨髓间充质干细胞移植后能够抑制神经细胞的凋亡。大量动物实验结果证明,骨髓间充质干细胞移植治疗脊髓损伤的临床应用前景是广阔的。     

骨髓间充质干细胞立体定向移植治疗大鼠脊髓损伤*

摘要 背景：传统观念认为,神经组织损伤后几乎不能再生,以往对SCI的治疗缺乏有效手段,致使本病致残率高,疗效差。干细胞治疗关键在于移植具有再生能力的干细胞,通过多种作用机制,可以重建中枢神经系统的结构和功能,近年来引起了广泛的关注。 目的：探讨立体定向移植骨髓间充质干细胞（MSCs）对大鼠脊髓损伤修复的影响并探讨其机制 设计、时间及地点：随机对照动物实验,于2007-10/2008-6在天津市环湖医院完成。 材料：1月龄SD大鼠20只,用于制备骨髓间充质干细胞;健康成年Wistar大鼠45只,雌性、同系,体质量280±20 g。将动物随机分为对照组、假手术组与移植组,每组各15只。 方法：密度梯度离心法结合贴壁筛选法分离骨髓间充质干细胞,经流式细胞仪鉴定为MSCs。以动脉瘤夹夹闭法制备大鼠脊髓损伤（SCI）模型,在SCI大鼠致伤后第7天,通过立体定向途径移植MSCs到移植组大鼠脊髓损伤中心,移植等量生理盐水至假手术组大鼠脊髓损伤中心,对照组大鼠不做处理。 主要观察指标：SCI大鼠损伤前及损伤后第7天、14天、30天、60天、90天的BBB评分;损伤后第90天处死大鼠,观察其脊髓组织中有无BrdU阳性细胞、Brdu+NSE、Brdu+GFAP、Brdu+bFGF、Brdu+BDNF免疫组化双染阳性细胞并观察NSE、GFAP、bFGF、BDNF单染阳性细胞。 结果： ①BBB评分发现,MSCs移植组大鼠BBB后肢功能评分恢复优于对照组（p<0.05）;假手术组BBB评分在损伤后30天内恢复速度慢于对照组（p<0.05）,至第90天与对照组比较无显著差异（P>0.05）;②免疫组织化学染色发现,移植组大鼠脊髓内在损伤中心及头、尾端距离脊髓损伤中心1cm处均可见BrdU染色阳性细胞及Brdu+NSE、Brdu+GFAP、Brdu+bFGF、Brdu+BDNF免疫组化双染阳性细胞。移植组NSE、GFAP、bFGF、BDNF单染阳性细胞数明显高于对照组和假手术组（p<0.05）。 结论： MSCs移植可以促进SCI大鼠的神经功能的恢复,其机制可能与移植细胞分化为神经元样和神经胶质细胞样细胞,并分泌或促进宿主分泌神经营养因子有关。 关键词 脊髓损伤 骨髓间充质干细胞 立体定向 细胞移植     

脐带源与骨髓源干细胞移植治疗大鼠脊髓损伤的疗效比较

目的 观察人脐带间充质干细胞(HUCMSCs)与大鼠骨髓间充质干细胞(BMSCs)移植治疗大鼠脊髓损伤后的功能恢复和急慢性排斥反应.方法 选取SD大鼠40只按随机数字表随机分成3组:脐带间充质干细胞移植组16只、骨髓间充质干细胞移植组16只和PBS组8只.采用脊髓半切手术制作大鼠脊髓损伤模型,损伤后对脐带间充质干细胞移植组进行HUCMSCs移植,对骨髓间充质干细胞移植组行大鼠BMSCs移植,对PBS组移植同等剂量的PBS.通过BBB评分对各组动物的术后恢复进行评估比较.用ELISA法测定移植后3组动物不同时间点(移植后1周、1个月、2个月及3个月)血中IL-2及IL-10的水平,并通过免疫组化的方法检测移植局部病理切片CD3+细胞浸润的情况.结果 在移植后1周、1个月、2个月及3个月时,检测到干细胞移植组各个时间点大鼠静脉血中IL-2水平较PBS组明显升高,而IL-10水平较PBS组明显下降,差异均有统计学意义(P＜0.05).大鼠骨髓间充质干细胞移植组在移植后1周、1个月、2个月及3个月时大鼠静脉血中IL-2水平较脐带间充质干细胞移植组明显升高,IL-10水平较对照组明显下降,差异均有统计学意义(P＜0.05).三组在急性期(细胞移植后l周)移植局部均有大量CD3+细胞浸润.而移植的慢性期(细胞移植后3个月)大鼠骨髓间充质干细胞移植组仍存在大量CD3+细胞浸润,与其他两组相比差异均有统计学意义(P＜0.05).结论 异体干细胞细胞移植后可发生急、慢性免疫排斥反应.HUCMSCs较大鼠BMSCs有更低的免疫源性,更利于脊髓损伤的功能恢复.     

Bone marrow-derived mesenchymal stem cells expressing the bFGF transgene promote axon regeneration and functional recovery after spinal cord injury in rats

Abstract

<title/>

Objective: To investigate neurological effects of transplanting bone marrow-derived mesenchymal stem cells (BMSCs) transfected with the basic fibroblast growth factor (bFGF) gene in spinal cord-injured rats.

Methods: Ninety-six male adult Sprague-Dawley rats were randomized into four groups: (1) pcDNA3·1-bFGF group; (2) pcDNA3·1 group; (3) BMSCs group; and (4) vehicle control (DMEM) group. After the rat model of acute spinal cord injury (SCI) was established, 1×10⁶ BMSCs or cells transfected with pcDNA3·1-bFGF or pcDNA3·1 were injected into rats of groups 1-3. At days 1, 7, 14, and 21 after injection, the Basso-Beattie-Bresnahan (BBB) locomotor rating scale was used to evaluate recovery of motor function. Expression changes of bFGF, myelin basic protein (MBP), and NF200 were examined by immunohistochemistry.

Results: The BBB score of DMEM group was significantly lower than those of groups 1-3 (P<0·05), but the score of pcDNA3·1-bFGF group was significantly higher than that of BMSCs group or pcDNA3·1 group at day 14 or 21 after injection (P<0·01). The number of bFGF-positive neurons in rats of pcDNA3·1-bFGF group was significantly higher than those of groups 1-3 at any time point (P<0·05). The optical density values of NF200-positive neurons and MBP-positive MBP axons in rats of pcDNA3·1-bFGF group were significantly higher than those of groups 1-3 at day 7 or 14 after injection (P<0·05).

Conclusions: bFGF gene-modified BMSCs not only effectively promoted axonal outgrowth but also enhanced recovery of neurological function after SCI in rats, and may be a good candidate to evaluate gene therapy of SCI in man.     

Review of transplantation of neural stem/progenitor cells for spinal cord injury

Spinal cord injury (SCI) is a debilitating condition often resulting in paralysis, yet currently there is no effective treatment. Stem cell transplantation is a promising therapeutic strategy for promoting tissue repair after SCI. Stem cells offer a renewable source of cells with inherent plasticity for tissue regeneration. Neural stem/progenitor cells (NSPCs) are multipotent cells that self-renew and are committed to the neural lineage, and thus, they are especially suited to SCI repair. NSPCs may differentiate into neural cells after transplantation into the injured spinal cord, replacing lost or damaged cells, providing trophic support, restoring connectivity, and facilitating regeneration. Here, we review experimental studies and considerations for clinical translation of NSPC transplantation for SCI.     

人脐带间充质干细胞移植治疗大鼠创伤性脑损伤

背景：脐带间充质干细胞体内移植治疗脑损伤的效果目前尚较少见报道。 目的：观察人脐带间充质干细胞移植对大鼠液压冲击脑损伤的治疗作用。 方法：从新生儿脐带中分离、培养间充质干细胞。制作中度打击大鼠脑损伤模型。实验分为4组：①脐带间充质干细胞移植组：损伤后原位移植脐带间充质干细胞。②对照组：损伤后原位注射等量DMEN/F12培养基。③单纯损伤组：仅施行损伤。④假损伤组：仅切开头皮及颅骨,不实施机械性损伤。 结果与结论：脐带间充质干细胞移植后1~3周,动物神经功能评分较对照组明显改善;4周后,各组动物神经功能评分均恢复正常。免疫组织化学检测表明少部分移植细胞表达神经元特异性烯醇化酶,胶质纤维酸性蛋白。与对照组相比,移植组损伤区血管内皮生长因子表达明显增加,凋亡细胞减少。提示脐带充间质干细胞脑内移植有助于促进创伤性脑损伤后的早期功能恢复,这种治疗效果是通过刺激宿主细胞分泌血管内皮生长因子,增加损伤区微血管密度,抑制宿主细胞凋亡等实现的。     

Functional recovery and microenvironmental alterations in a rat model of spinal cord injury following human umbilical cord blood-derived mesenchymal stem cells transplantation

BACKGROUND: Transplantation of human umbilical cord blood-derived mesenchymal stem cells (MSCs) has been shown to benefit spinal cord injury (SCI) repair. However, mechanisms of microenvironmental regulation during differentiation of transplanted MSCs remain poorly understood. OBJECTIVE: To observe changes in nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and interleukin-8 (IL-8) expression following transplantation of human umbilical cord-derived MSCs, and to explore the association between microenvironment and neural functional recovery following MSCs transplantation.DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Department of Orthopedics, First Affiliated Hospital of Soochow University from April 2005 to March 2007. MATERIALS: Human cord blood samples were provided by the Department of Gynecology and Obstetrics, First Affiliated Hospital of Soochow University. Written informed consent was obtained. METHODS: A total of 62 Wister rats were randomly assigned to control (n = 18), model (n = 22, SCI + PBS), and transplantation (n = 22, SCI + MSCs) groups. The rat SCI model was established using the weight compression method. MSCs were isolated from human umbilical cord blood and cultured in vitro for several passages. 5-bromodeoxyuridine (BrdU)-labeled MSCs (24 hours before injection) were intravascularly transplanted. MAIN OUTCOME MEASURES: The rats were evaluated using the Basso, Beattie and Bresnahan (BBB) locomotor score and inclined plane tests. Transplanted cells were analyzed following immunohistochemistry. Enzyme-linked immunosorbant assay was performed to determine NGF, BDNF, and IL-8 levels prior to and after cell transplantation.RESULTS: A large number of BrdU-positive MSCs were observed in the SCI region of the transplantation group, and MSCs were evenly distributed in injured spinal cord tissue 1 week after transplantation. BBB score and inclined plane test results revealed significant functional improvement in the transplantation group compared to the model group (P< 0.05), which was maintained for 2-3 weeks. Compared to the model group, NGF and BDNF levels were significantly increased in the injured region following MSCs transplantation at 3 weeks (P < 0.05), but IL-8 levels remained unchanged (P > 0.05).CONCLUSION: MSCs transplantation increased NGF and BDNF expression in injured spinal cord tissue. MSCs could promote neurological function recovery in SCI rats by upregulating NGF expression and improving regional microenvironments.     

Transplantation of human umbilical cord blood mesenchymal stem cells to treat a rat model of traumatic brain injury

In the present study, human umbilical cord blood mesenchymal stem cells were injected into a rat model of traumatic brain injury via the tail vein. Results showed that 5-bromodeoxyuridine-labeled cells aggregated around the injury site, surviving up to 4 weeks post-transplantation. In addition, transplantation-related death did not occur, and neurological functions significantly improved. Histological detection revealed attenuated pathological injury in rat brain tissues following human umbilical cord blood mesenchymal stem cell transplantation. In addition, the number of apoptotic cells decreased. Immunohistochemistry and in situ hybridization showed increased expression of brain-derived neurotrophic factor, nerve growth factor, basic fibroblast growth factor, and vascular endothelial growth factor, along with increased microvessel density in surrounding areas of brain injury. Results demonstrated migration of transplanted human umbilical cord blood mesenchymal stem cells into the lesioned boundary zone of rats, as well as increased angiogenesis and expression of related neurotrophic factors in the lesioned boundary zone.