绿色荧光蛋白转基因大鼠神经干细胞体外分化与体内移植的实验研究

目的 探讨绿色荧光蛋白（green fluorescent protein，GFP）转基因大鼠胚胎脊髓神经干细胞（neural stem cells，NSC）体外增殖、分化的生物学活性以及植入体内后存活、分化和迁移的情况。方法 对GFP转基因大鼠的胚胎脊髓NSC进行体外分离、培养和诱导分化，并应用特异性抗体分别对神经干细胞、神经元、星形胶质细胞和少突胶质细胞进行免疫荧光染色鉴定。建立F344大鼠胫神经切断的动物模型，将体外稳定传代的GFP-NSC单细胞悬液移植于胫神经远侧段。移植12周后取材，应用激光共聚焦显微镜和普通荧光显微镜观察神经干细胞体内的存活、分化和迁移情况，并对胫神经冰冻切片行神经元特异性免疫荧光染色鉴定。结果 通过体外分离培养的方法获得了稳定传代的GFP-NSC，体外诱导分化形成神经元、星形胶质细胞和少突胶质细胞。GFP-NSC体内移植后，部分分化为神经元，并发出轴突样的结构向远端生长。结论 GFP-NSC具有良好的生物学活性，体内移植后可以分化为神经元，为进一步研究其体内移植防治骨骼肌失神经萎缩及治疗其他神经元损伤性疾病提供了实验依据。     

神经干细胞移植治疗结肠去神经节小鼠

目的 观察神经干细胞在去神经节小鼠结肠壁内的存活分化,探讨神经干细胞移植治疗结肠无神经节细胞症的可行性.方法 0.5%苯扎氯铵(BAC)处理8周龄昆明小鼠结肠浆膜层选择性去除结肠肇神经节制作巨结肠模型,原代培养新生小鼠大脑皮质来源神经干细胞,Hoechsd3342标记传代纯化后神经干细胞.运用微量注射器将标记后的神经干细胞移植入模型鼠病变肠段,分别于术后1、2、3、4周行大体观察,苏木素-伊红(HE)染色,免疫组织荧光检测小鼠生物学特性和神经干细胞存活分化情况.结果 原代培养神经干细胞Nestin表达阳性,体外培养可分化为神经元和神经胶质细胞;BAC处理后,HE染色及免疫组织化学染色显示小鼠结肠肌间及黏膜下神经节消失;神经干细胞移植后各观测时间点可见荧光标记细胞,免疫组织荧光检测显示术后1周结肠壁存在巢蛋白(Nestin)表达阳性细胞,3周后可见神经元特异性烯醇化酶(NSE)及胶质纤维酸性蛋白(GFAP)表达阳性细胞,对照组未观察到阳性表达.结论 神经干细胞可以在去神经节小鼠结肠肇内存活并分化为神经元及胶质细胞,部分恢复肠道神经的调节作用.     

神经干细胞移植在神经系统疾病治疗中的应用

神经干细胞(NSC)来源于胚胎或者成体,能自我更新,可以分化为神经元、星形胶质细胞和少突胶质细胞等,具有低免疫原性.NSC在体外能大量增殖,移植后在神经系统长期存活,并能分化整合到宿主中枢神经系统中.     

超顺磁氧化铁、增强型绿色荧光蛋白双标中脑神经干细胞移植治疗帕金森病

目的 观察超顺磁氧化铁(SPIO)、增强型绿色荧光蛋白(EGFP)双标中脑神经干细胞(mNSCs)移植对帕金森病(PD)大鼠的治疗作用.方法 6-羟多巴胺立体定向注射建立PD大鼠模型,随机分为对照组(n=12)和细胞移植组(n=12).将SPIO、EGFP双标mNSCs移植到PD大鼠纹状体区.阿朴吗啡(APO)诱导PD大鼠旋转行为评估细胞移植的治疗作用.磁共振成像动态观察移植细胞的存活和迁移,免疫荧光组织化学研究移植细胞的存活、迁移和分化.结果 与对照组比较,SPIO、EGFP双标mNSCs移植能显著改善APO诱导PD大鼠的异常旋转行为(P＜0.01);大多数移植细胞停留于移植原位,仅少数细胞向周围脑组织迁移;移植8周后,大多数mNSCs保持其未分化状态(37±6)%或分化为神经胶质细胞(36±4)%,仅少数细胞分化为DA神经元(6±2)%.结论 通过MRI成像可对体内移植的SPIO标记细胞进行活体示踪.SPIO、EGFP双标mNSCs移植可显著改善PD大鼠的运动障碍.     

脐血间充质干细胞移植对大鼠局灶性脑缺血的影响

目的　从人脐血中分离纯化间充质干细胞(MSC) ,观察其移植对大鼠大脑中动脉栓塞后神经功能恢复的影响及细胞的存活、迁移向神经细胞分化的情况。方法　雄性SD大鼠45只,用线栓法建立大鼠大脑中动脉栓塞(MCAO)模型,大鼠随机分为3组:MSC移植组、单核细胞组和生理盐水组。移植后1、7、14、2 1、2 8d采用改良神经功能损害评分(mNSS)观察大鼠神经功能恢复情况,应用免疫组织化学和免疫荧光双标记技术检测5溴 2脱氧尿核苷(BrdU )标记的MSC细胞的存活、迁移及其胶质纤维酸性蛋白(GFAP)和神经元特异性核蛋白(NeuN)的表达。结果　人脐血MSC细胞移植可显著提高大鼠局灶性脑缺血后神经功能的恢复(P <0 .0 5 )。移植的MSC细胞可在大鼠脑组织中存活,并向缺血区域迁移,11.67%MSC细胞表达GFAP ,3 .72 %MSC细胞表达NeuN。结论　人脐血中含有MSC细胞并可促进局灶性脑缺血大鼠的神经功能恢复,移植细胞可在大鼠脑缺血区域中存活、迁移并向星形胶质细胞或神经元分化     

人胚胎神经干细胞延迟移植对脊髓损伤大鼠运动功能的影响及其机制

目的:初步探讨人胚胎神经干细胞(human fetal neural stem cells,hfNSCs)延迟移植对脊髓损伤大鼠运动功能的影响及其机制。方法:体外分离、培养和鉴定hfNSCs,选取200~250g的Wistar大鼠28只建立T9~T11节段脊髓挫伤模型,损伤后第9天取20只BBB评分为4~5分的大鼠随机分为对照组及移植组(每组10只),同时在T10水平脊髓中线两侧1.5mm处分别给予2μl不含hfNSCs的杜氏磷酸缓冲液(DPBS)及2μl含有105个体外培养第2代hfNSCs的DPBS,细胞移植后第7、14、28、42、56天用BBB评分评价两组后肢运动功能,移植后第28天及第56天用免疫组织化学法检测移植组大鼠损伤脊髓局部神经胶质纤维酸性蛋白(GFAP)、微管相关蛋白2(MAP2)和2′,3′-环腺苷酸-3′-磷酸二酯酶(CNPase)的表达情况,以评价大鼠损伤脊髓局部hfNSCs的存活及分化情况。移植后第56天髓磷脂碱性蛋白(MBP)染色及透射电子显微镜扫描观察移植组与对照组损伤局部髓鞘形成情况。结果:体外培养的hfNSCs表达Nestin,并可分化为星形胶质细胞[(61.2±1.6)%]、神经元[(27.6±3.4)%]及少突胶质细胞[(6.0±5.5)%]。移植前及移植后第7、14天两组BBB评分比较无统计学差异(P>0.05),移植组在移植后第28、42、56天评分(分别为8.30±1.07分,11.30±1.05分,15.10±1.12分)较对照组(分别为7.00±0.98分,8.30±1.02分,9.10±0.96分)明显提高(P<0.05)。在移植后第28天hfNSCs可分化为GFAP阳性的星形胶质细胞、MAP2阳性的神经元和CNPase阳性的少突胶质细胞,在移植后第56天hfNSCs仍在损伤局部存活并可分化为星形胶质细胞和少突胶质细胞,但未见神经元分化;移植后第56天星形胶质细胞的积分光密度(IOD)值(1502.31±131.92)大于移植后第28天(628.98±119.31)(P<0.05),而少突胶质细胞在移植后第28天及第56天的变化无统计学差异。移植后第56天MBP染色证实人源性少突胶质细胞参与损伤区髓鞘的形成,透射电子显微镜扫描结果显示移植组有完整和连续的髓鞘形成,对照组髓鞘呈断裂及畸形表现。结论:hfNSCs延迟移植可促进脊髓损伤大鼠运动功能的恢复,其机制可能与促进损伤局部髓鞘的形成有关。     

经颈动脉注入神经干细胞治疗脑出血后遗症的时间窗及疗效

目的 探讨经颈动脉神经干细胞(NSCs)移植治疗大鼠脑出血(ICH)后遗症的适宜移植时间窗和疗效.方法 48只Wistar大鼠脑出血模型,随机分为实验组和对照组.分别于脑出血后第2、7、14、21、28天经颈动脉行5-溴脱氧尿嘧啶核苷(BrdU)体外标记的神经干细胞移植.对照组大鼠,仅接受颈动脉内注入等量DMEM培养液.每周测试大鼠行为功能评分;2个月后处死所有动物,行组织化学染色,观察神经干细胞在脑内的分布、分化,评定病灶体积大小.结果 与对照组比较,NSCs移植组大鼠行为功能改善明显(ANOVA,P＜0.05);脑出血第7天移植组在移植后第3周和ICH后第9周的行为功能评分显著优于其他时间移植组(SNK,P＜0.05).ICH后第7天和第14天移植组BrdU阳性细胞计数显著多于ICH后第2、21和第28天移植组.ICH后第2天移植的NSCs绝大部分分化为星形胶质细胞(84.5±7.6)%;而ICH后第21和28天移植的NSCs分化为神经元的比例明显增大(35.4±3.1)%、(37.2±4.1)%;然而,7～14 d移植组,分化为神经元的绝对数量显著高于其他治疗组(P＜0.01).结论 经皮颈动脉注射是一种可行的、微侵袭和高效的细胞移植方法.在脑出血后7～14 d期间进行神经干细胞移植,能显著提高细胞存活率和移植细胞分化为神经元,明显促进功能改善.     

骨髓干细胞移植和动员对重症急性胰腺炎肾损伤的保护作用

目的 探讨骨髓间充质干细胞(MSC)移植和动员对重症急性胰腺炎(SAP)大鼠急性肾损伤的保护作用.方法 240只SD大鼠按照随机数字表法分为假手术组、模型组、干细胞移植组、干细胞动员组和联合组,每组48只.腹腔注射L-精氨酸制作SAP大鼠模型.假手术组在制作SAP大鼠模型后腹腔注射生理盐水,干细胞移植组制作SAP大鼠模型后6h经股静脉注入自体MSC 1.2 ml,干细胞动员组制作SAP大鼠模型前连续3d皮下注入重组人粒细胞集落刺激因子(G-CSF) 40 μg/kg,联合组则联合应用MSC和G-CSF.各组大鼠再按术后不同时相点分为12、24、48、72 h亚组,每组12只.在术后相应时相点观察各组大鼠的存活情况,肾脏组织的病理变化,肾小管上皮细胞Bax和Bcl-2蛋白的表达情况和细胞凋亡指数,检测血清中TNF-α、IL-6、血清尿素氮(BUN)、肌酐(Cr)、LDH、C反应蛋白(CRP)的含量.采用单因素方差分析各组间的指标,两两比较用SNK-q检验,大鼠存活情况用Fisher确切概率法.结果 假手术组大鼠全部存活.模型组大鼠术后48、72 h分别存活11只和8只.干细胞移植组、干细胞动员组和联合组术后48 h前未见大鼠死亡,术后72 h分别存活11、10和11只,与模型组比较,差异无统计学意义(P＞0.05).各治疗组术后肾脏组织病理变化均较模型组减轻,联合组损伤减轻最为明显.术后12 ～72 h肾小管上皮细胞Bax蛋白、Bcl-2蛋白、肾小管细胞凋亡指数变化情况:模型组分别为12.80±1.78 ～20.30±2.40、4.34±1.20 ～3.03±1.06、12.65％±2.31％ ～35.10％±5.54％;干细胞移植组分别为9.68±2.11～17.01±2.54、5.57±1.35～4.13±1.05、6.20％±1.53％ ～ 17.50％±2.80％;干细胞动员组分别为10.05±2.17～16.81±2.55、5.49±1.48～4.19±1.05、6.41％±1.64％ ～ 17.14％±2.27％;联合组分别为8.33±2.06～14.03±2.27、6.60±2.11 ～5.63±1.52、5.80％±1.52％～12.30％±2.43％.联合组术后24、72 h Bax蛋白,48、72 h Bc1-2蛋白,24、48、72 h凋亡指数与干细胞移植组和干细胞动员组比较,差异有统计学意义(P＜0.05);干细胞移植组与干细胞动员组比较,差异无统计学意义(P＞0.05);各治疗组术后12 ～72 h炎症因子及肾功能指标较模型组不同程度降低,以联合组最明显.联合组术后72 hTNF-α含量,48、72 h IL-6含量,48、72 h BUN含量,48、72 h Cr含量,24、48、72 h LDH含量,72 h CRP含量与干细胞移植组和干细胞动员组比较,差异有统计学意义(P＜0.05);干细胞移植组与于细胞动员组比较,差异无统计学意义(P＞0.05).结论 自体MSC移植与动员能有效减轻SPA大鼠的肾损伤,可能与MSC参与组织的病理再生修复、抗炎症及抑制细胞凋亡等机制有关.     

尿嘧啶脱氧核苷及超顺磁性氧化铁双标记的神经干细胞活体移植后细胞迁徙及功能性分化的实验研究

目的观察尿嘧啶脱氧核苷(Brdu)及超顺磁性氧化铁(SPIO)双标记的神经干细胞活体移植后存活细胞的迁徙及分化。方法SPIO和Brdu对神经干细胞进行双标记,移植至大脑中动脉梗死模型的对侧脑组织,术后采用MRI监测。第6周MRI检查后取组织切片行免疫组织化学染色,观察神经干细胞的迁徙及分化。结果双标神经干细胞活体移植后第3周MRI开始显示细胞沿胼胝体迁徙,第6周脑组织免疫组织化学染色亦显示干细胞沿胼胝体向对侧迁徙,免疫组织化学荧光双标染色显示移植的神经干细胞可以分化为星形胶质细胞及神经元。结论移植存活的神经干细胞在迁徙过程中能够进行功能性分化。     

神经营养因子-3修饰的神经干细胞移植大鼠受损脊髓后的存活及分化

目的 观察增强型绿色荧光蛋白(EGFP)标记的神经营养因子(NT)-3修饰的胚胎脊髓来源的神经干细胞(NSCs)移植入受损脊髓后的存活及分化.方法 将体外构建的EGFP标记NT-3修饰的NSCs(NT-3-NSCs)移植入SD在胸9水平脊髓半切的大鼠(10只),通过免疫组织化学方法检测移植细胞的存活、分化及NT-3的表达,并与未进行NT-3修饰的NSCs移植组(NSCs)(10只)进行比较.结果 NT-3-NSCs组移植细胞存活数(304±40)比NSCs组(258±41)更多(P>0.05).NT-3-NSCs组中NT-3阳性细胞的比例(74.2±14.1)%明显高于NSCs组(22.9±11.1)%(P<0.01);NT-3-NSCs组中少突胶质细胞分化率(53.8±12.4)%明显高于NSCs组(39.7±13.7)%(P<0.05).结论 NT-3修饰能促进NSCs在受损脊髓内存活、表达NT-3及分化为少突胶质细胞,有助于NSCs移植对脊髓损伤(SCI)的治疗效果.     

神经干细胞移植改善脑缺血大鼠的神经功能研究

目的 探讨大鼠神经干细胞移植改善脑缺血所致神经功能损伤的可行性。方法 制作大鼠大脑中动脉夹闭致脑缺血模型，记录并比较损伤和移植干细胞前后大鼠的神经功能。6周后杀死大鼠，研究干细胞在体内分化和迁移的情况。结果 接受干细胞移植大鼠神经功能的改善显著好于未移植干细胞的对照组。免疫组织化学方法证实移植后干细胞在脑内分化成胶质细胞和少量的神经细胞，并向损伤区域迁移。结论 神经干细胞体内外均具有多向分化潜能。神经干细胞移植能够有效改善脑缺血大鼠的神经功能。     

Fetal cortical cells survive in focal cerebral infarct after permanent occlusion of the middle cerebral artery in adult rats.

Fetal rat cortical cells have been shown previously to survive at the periphery of cerebral infarction. The present study was designed to examine the ability of fetal cells to survive at the edge of the central core of ischemia. In three groups of 8 adult Sprague-Dawley rats, fetal cortical cells from ED 16 were stereotactically transplanted at 3 h, 24 h, and 7 days after unilateral middle cerebral artery occlusion. In 6 rats, fetal cells were transplanted by using the same coordinates, without arterial occlusion, for control. In the ischemic groups, overall graft survival was 85%, and in the control group, all grafts survived. Graft survival was determined by light microscopy. No significant difference was found in the survival of grafts transplanted at different intervals after middle cerebral artery occlusion. It is concluded that fetal cortical cells can survive in cerebral tissue undergoing severe ischemic change.     

脂肪干细胞侧脑室定向移植改善脑出血大鼠的神经功能

目的 研究脂肪干细胞(ADSC)侧脑室定向移植改善脑出血大鼠的神经功能及其作用机制.方法 体外培养大鼠ADSC,于移植前48 h用Brdu抗原标记.另取大鼠制备尾状核脑出血(ICH)模型,将建模后的大鼠分为2组,ADSC组于建模后48 h定向右侧脑室移植标记的ADSC,对照组输注等体积生理盐水.分别在完成ICH建模时及移植后1、3、7、14、28 d等时间点,对两组大鼠进行神经行为学评分,采用免疫荧光染色法检测Brdu阳性细胞及神经元和星形胶质细胞的分化情况,按照原位末端标记法(TUNEL)试剂盒的方法检测细胞凋亡情况,按照血管内皮细胞生长因子(VEGF)试剂盒说明检测VEGF的表达及血管新生情况.结果 体外ADSC具有分化为成脂肪细胞和成骨细胞的能力.在移植后3、7、14 d,ADSC组大鼠神经行为学评分明显优于对照组(P＜0.05).免疫双荧光检测显示,Brdu标记的ADSC在大鼠脑组织内存活并且向病灶部位迁移,部分分化为神经胶质细胞和神经元细胞.TUNEL检测显示,移植后3 d时ADSC组凋亡细胞数目明显少于对照组(P＜0.05).移植3 d后,ADSC组VEGF的表达均明显高于对照组(P＜0.05).结论 经侧脑室定向移植的ADSC在脑损伤病灶周围可以存活,并可分化为神经样细胞;移植后ADSC可抑制细胞凋亡,分泌VEGF促进新生血管的生成,从而改善大鼠的神经功能.

Abstract：

Objective To study improvement of neural function by stereotaxic transplantation of adipose-derived stem cells (ADSC) into lateral cerebral ventricle after intracerebral hemorrhage in rats and its mechanism. Methods ADSC were cultured and proliferated in vitro, which had been marked with Brdu for 48 h before transplantation. The rat caudate nucleus hemorrhage (ICH) models were divided into 2 groups. ADSC were stereotaxically transplanted into the right lateral ventricles in ADSC group, and equal volume of saline was transplanted into control group. The score of neurological behavior were evaluated at modeling and 1, 3, 7, 14, 28 days after transplantation respectively.Double-staining immunofluorescence technique was used to detect Brdu-positive cells and the differentiation of neurons and astrocytes. In accordance with the instructions of TUNEL kit, cell apoptosis, and the expression of VEGF and angiogenesis were assayed. Results In vitro ADSC expressed undergo osteogenic and adipogenic differentiation. Compared with the control group, ADSC group had better motor function at 3, 7, and 14 days (P＜0. 05). Double-staining immunofluorescence showed mostly grafted Brdu-reactive ADSC had migrated to the hematoma zone, and some survivedand expressed Neun of Gfap. TUNEL analysis revealed that, 3 days after transplantation, the number of apoptotic cells in ADSC group was significantly less than in the control group (P＜0. 05). Three days after transplantation, VEGF expression levels in ADSC group were significantly higher than in the control group (P＜0. 05). Conclusion ADSC stereotaxially transplanted into the lateral ventricle can survive and differentiate into neuron-like cells. ADSC transplantation may reduce apoptosis and secret VEGF to promote the angiogenesis, and improve neural functional in intracerebral hemorrhage rats.     

胚胎干细胞移植修复脊髓损伤的实验研究

目的观察胚胎干细胞（embryonic stem cell,ES）诱导的神经前体细胞移植,对小鼠脊髓损伤神经功能恢复的影响。方法取由上海市发育生物学重点实验室提供的ES进行细胞培养和体外诱导,收集ES衍生细胞。并进行RT-PCR检测。将50只C57/BL6J小鼠制备为T9、10脊髓半横断模型,将存活的28只小鼠随机分为三组。假手术组（A组）：9只,未作任何处理;手术/细胞组（B组）：10只,于距损伤区域以远约1cm的椎管内注射2~3μl制备的ES衍生细胞,总细胞数为9×105个;手术/DMEM组（C组）：9只,按B组方法注射2~3μl DMEM。术后1、2、4、6和8周采用BBB后肢功能评分观察小鼠神经功能恢复情况,取损伤脊髓进行X-gal染色和免疫组织化学染色观察。结果ES经体外诱导培养,呈圆形或椭圆形小集落生长,有1个或多个核仁。RT-PCR检测,ES细胞诱导后表达巢蛋白及微管相关蛋白,但未表达胶质纤维酸性蛋白。小鼠实验,BBB后肢功能评分显示术后各时间点A组与B、C组比较,差异均有统计学意义（P〈0.01）。B组与C组比较,1、2和4周时,差异有统计学意义（P〈0.01）;6、8周时,组间差异无统计学意义（P〉0.05）。X-gal染色观察,B组呈阳性染色,A、C组均为阴性。免疫组织化学染色观察,B组在损伤脊髓部位,表达兔抗神经微丝蛋白,未表达胶质纤维酸性蛋白。结论将ES培养诱导分化为神经前体细胞移植后,能够存活、迁移,并分化为神经元,但未明显改善神经功能。     

Embryonic stem cells differentiated in vitro as a novel source of cells for transplantation

The controlled differentiation of mouse embryonic stem (ES) cells into near homogeneous populations of both neurons and skeletal muscle cells that can survive and function in vivo after transplantation is reported. We show that treatment of pluripotent ES cells with retinoic acid (RA) and dimethylsulfoxide (DMSO) induce differentiation of these cells into highly enriched populations of γ-aminobutyric acid (GABA) expressing neurons and skeletal myoblasts, respectively. For neuronal differentiation, RA alone is sufficient to induce ES cells to differentiate into neuronal cells that show properties of postmitotic neurons both in vitro and in vivo. In vivo function of RA-induced neuronal cells was demonstrated by transplantation into the quinolinic acid lesioned striatum of rats (a rat model for Huntington's disease), where cells integrated and survived for up to 6 wk. The response of embryonic stem cells to DMSO to form muscle was less dramatic than that observed for RA. DMSO-induced ES cells formed mixed populations of muscle cells composed of cardiac, smooth, and skeletal muscle instead of homogeneous populations of a single muscle cell type. To determine whether the response of ES cells to DMSO induction could be further controlled, ES cells were stably transfected with a gene coding for the muscle-specific regulatory factor, MyoD. When induced with DMSO, ES cells constitutively expressing high levels of MyoD differentiated exclusively into skeletal myoblasts (no cardiac or smooth muscle cells) that fused to form myotubes capable of spontaneous contraction. Thus, the specific muscle cell type formed was controlled by the expression of MyoD. These results provided evidence that the specific cell type formed (whether it be muscle, neuronal, or other cell types) can be controlled in vitro. Further, these results demonstrated that ES cells can provide a source of multiple differentiated cell types that can be used for transplantation.     

Transplantation of adult rat spinal cord stem/progenitor cells for spinal cord injury

Stem/progenitor cells derived from the ependymal region of the spinal cord have the ability to self-renew and are multipotential for neurons and glia. These cells may have the ability to regenerate the injured mammalian spinal cord as they do in some lower vertebrates. However, the optimal conditions for transplantation and the fate of transplanted cells are not fully known. In the current study, spinal cord stem/progenitor cells were cultured from adult male rats expressing enhanced green fluorescent protein (eGFP). Neurospheres were transplanted at the time of clip compression injury (35-g force) into the injury site, or 1 mm rostral and caudal to the injury site. Neurospheres were also transplanted into a subacute model (day 9 after injury) and a chronic model (day 28 after injury). Functional recovery was also studied in an acute injury model with weekly locomotor testing over a 16-week period. A significant increase in cell survival at 7 days was seen in rats receiving rostral and caudal injections as compared to injection directly into the site of injury. A significant increase in cell survival was also seen in rats receiving subacute transplants at 9 days after injury. Transplanted cells differentiated primarily into astrocytes (31.2%) and oligodendrocytes (50.3%), and a small number of neurons (1%). No improvement was seen in the Basso, Beattie and Bresnahan (BBB) locomotor rating scale after acute transplantation as compared with injury only, although surviving transplanted cells were identified that had migrated across the injury site from the rostral and caudal injection sites.     

臂丛损伤神经干细胞脊髓前角移植后分化情况及对运动神经元的保护作用

目的观察臂丛根性撕脱伤后将脊髓源性神经干细胞(neuralstemcell,NSC)移植于脊髓前角后的存活、分化情况及对脊髓前角受损运动神经元的保护作用。方法取新生鼠脊髓,分离获得脊髓源性神经干细胞,体外培养、扩增、鉴定、5溴2-脱氧尿苷(BrdU)标记。取SD大鼠60只,随机分成实验组、对照组和单纯组。从后路制备C5~C7臂丛神经根性撕脱伤动物模型。实验组移植神经干细胞于C6脊髓前角,对照组移植灭活神经干细胞,单纯组不作移植。术后1、2、4、8、12周取脊髓标本进行组织学与免疫组化染色观察。结果神经干细胞移植入脊髓后能存活、分化;臂丛根性撕脱伤后脊髓前角运动神经元数目明显减少;实验组神经干细胞移植后2、4、8、12周各个时间点运动神经元的存活率均高于对照组和单纯组。结论臂丛根性撕脱伤脊髓前角神经干细胞移植后能存活并分化为神经元及星型胶质细胞,脊髓源性神经干细胞移植能明显减少前角运动神经元的继发性死亡,对脊髓前角受损运动神经元有保护作用。     

Delayed transplantation of human neurons following brain injury in rats: a long-term graft survival and behavior study

The NTera2 (NT2) cell line is a homogeneous population of cells, which, when treated in vitro with retinoic acid, terminally differentiate into postmitotic neuronal NT2N cells. Although NT2N neurons transplanted in the acute (24 h postinjury) period survive for up to 1 month following experimental traumatic brain injury (TBI), nothing is known of their ability to survive for longer periods or of their effects when engrafted during the chronic postinjury period. Adult male Sprague-Dawley rats (n = 348; 360-400 g) were initially anesthetized and subjected to severe lateral fluid-percussion (FP) brain injury or sham injury. At 1 month postinjury, only brain-injured animals showing severe neurobehavioral deficits received cryopreserved NT2N neurons stereotaxically transplanted into three sites in the peri-injured cortex (n = 18). Separate groups of similarly brain-injured rats received human fibroblast cells (n = 13) or cell suspension vehicle (n = 14). Sham-injured animals (no brain injury) served as controls and received NT2N transplants (n = 24). All animals received daily immunosuppression for three months. Behavioral testing was performed at 1, 4, 8, and 12 weeks post-transplantation, after which animals were sacrificed for histological analysis. Nissl staining and anti-human neuronal specific enolase (NSE) immunostaining revealed that NT2N neurons transplanted in the chronic post-injury period survived up to 12 weeks post-transplantation, extended processes into the host cortex and immunolabeled positively for synaptophysin. There were no statistical differences in cognitive or motor function among the transplanted brain-injured groups. Long-term graft survival suggests that NT2N neurons may be a viable source of neural cells for transplantation after TBI and also that these grafts can survive for a prolonged time and extend processes into the host cortex when transplanted in the chronic post-injury period following TBI.     

Intraarterial administration of marrow stromal cells in a rat model of traumatic brain injury.

To test the efficacy of various delivery routes of stem cells to treat cerebral injury, we investigated the parenchymal distribution of marrow stromal cells (MSCs) injected into the internal carotid artery (ICA) of the adult rat after traumatic brain injury (TBI). Bromodeoxyuridine (BrdU)-labeled MSCs were injected via the ipsilateral ICA at 24 h after TBI. Using histology and immunohistochemistry, the distribution of implanted MSCs was analyzed at 7 days after transplantation. Four groups (n = 4/group) were studied: group 1, animals transplanted with MSCs cultured with NGF and BDNF at 24 h after TBI; group 2, animals transplanted with MSCs cultured without NGF and BDNF; group 3, animals injected with a placebo, phosphate buffered saline into the ICA at 24 h after TBI; and group 4, rats subjected to TBI only. In groups 1 and 2, BrdU-positive cells were localized to the boundary zone of the lesion, corpus callosum and cortex of the ipsilateral hemisphere. The number of BrdU-positive cells was significantly higher in the ipsilateral hemisphere than in the contralateral hemisphere. More MSCs infused intraarterially engrafted in group 1 (18.9%) than in group 2 (14.4%, p < 0.05). Using double staining, BrdU-positive cells expressed MAP-2, NeuN, and GFAP in both groups 1 and 2, with this expression being greater in group 1 and the difference between two groups reaching statistical significance in case of MAP-2. Our data suggest that intraarterial transplantation of MSCs is a viable route for the intracerebral administration of MSCs for the treatment of TBI, since MSCs infused intraarterially after TBI survive and migrate into the brain. Some implanted MSCs express proteins specific to neurons and astrocytes. The addition of NGF and BDNF promote migration of MSCs into the brain and subsequent expression of neuronal protein MAP-2.