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。     

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。     

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     

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。     

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.     

咯利普兰对大鼠脊髓损伤后保护作用及其机制

目的探讨咯利普兰对大鼠脊髓损伤(SCI)后的保护作用及其可能机制。方法将84只SD大鼠随机分为三组:假手术组(n=4)、损伤组(n=40)和治疗组(n=40)。大鼠SCI模型采用纽约大学脊髓重物坠落伤模型。治疗组伤后即刻腹腔内注射咯利普兰,剂量为0.5 mg(/kg.d),2次/d,连续3d。损伤前后对损伤组和治疗组大鼠进行开放场地试验(BBB)评估大鼠脊髓功能。免疫组化染色分析三组大鼠损伤前后脊髓兴奋性氨基酸转运蛋白4(EAAT4)表达情况。结果损伤组和治疗组伤后即刻大鼠BBB评分均为0,处于完全瘫痪状态;然后BBB评分逐渐增高;伤后42、56和64d,治疗组BBB评分明显高于损伤组(P<0.01)。假手术组脊髓组织形态正常,EAAT4表达较少;脊髓损伤1周后,损伤中央出现较大空洞,EAAT4表达明显升高;而治疗组,组织空洞缩小,EAAT4表达较损伤组明显增加。结论咯利普兰有助于大鼠脊髓损伤后功能恢复,其机制可能与增加EAAT4表达有关。     

动脉移植BMSCs对大鼠缺血再灌注损伤脊髓CNTF和STAT3的影响

目的探讨肾下腹主动脉移植骨髓间充质干细胞(BMSCs)对大鼠缺血再灌注损伤脊髓CNTF和STAT3的影响及对损伤脊髓功能恢复的作用。方法 24只成年SD雌性大鼠随机分为假手术组、对照组及移植组3组,每组8只。术后对大鼠进行后肢神经行为学评分和运动诱发电位检测,采用RT-PCR、Western blot检测大鼠缺血节段脊髓内CNTF和STAT3的表达变化。结果与假手术组比较,对照组和移植组BBB评分于术后0 d、5 d、10 d、15 d显著降低(P<0.01),MEP潜伏期延长(P<0.01)、波幅减小(P<0.01),脊髓CNTF和STAT3 mRNA和蛋白表达增加(P<0.01);与对照组比较,移植组BBB评分于术后5¹5 d增高(P<0.01),MEP潜伏期缩短(P<0.01)、波幅增加(P<0.01),CNTF和STAT3 mRNA和蛋白表达增加(P<0.01)。结论肾下腹主动脉移植BMSC可能通过增加损伤脊髓局部CNTF和STAT-3表达促进脊髓缺血再灌注损伤大鼠后肢功能恢复。     

胎鼠神经干细胞移植对大鼠脊髓损伤后神经细胞凋亡及凋亡抑制基因Bcl-2表达的影响

目的研究胎鼠神经干细胞(NSCs)移植对大鼠脊髓损伤(SCI)后神经细胞凋亡及凋亡抑制基因Bcl-2表达的影响。方法 40只SD大鼠随机分为正常对照组(Normal组),脊髓损伤组(SCI组),神经干细胞组(NSC组),神经干细胞标记组(BrdU+NSCs组)。采用电控脊髓损伤打击装置制作模型,5-溴脱氧尿嘧啶核苷(Br-dU)法标记处于对数生长期的NSCs,SCI后即刻进行NSCs移植。免疫组化法观察BrdU标记NSCs的存活、迁移及凋亡抑制基因Bcl-2的表达,TUNEL法标记凋亡细胞(免疫组化及免疫荧光显色),改良Rivlin法观察大鼠后肢运动功能的恢复情况。结果 BrdU+NSCs组在损伤脊髓区域可检测到BrdU标记的阳性NSCs。BrdU+NSC组与NSC组各时间点凋亡阳性细胞数均比SCI组减少(P<0.01),Bcl-2免疫阳性细胞光密度值比SCI组明显增加(P<0.01),且Bcl-2表达高峰延长至伤后7d;移植后7d、14d、28d后肢运动功能评分较SCI组明显升高(P<0.01)。Br-dU+NSC组与NSC组之间比较无明显差异(P>0.05)。结论体外培养的胚胎大鼠NSCs可在脊髓损伤区域存活、迁移,并能通过上调Bcl-2的表达来抑制大鼠脊髓损伤后神经细胞的凋亡,从而促进大鼠瘫痪肢体功能的恢复。     

5000/自体骨髓干细胞动员移植与手术移植治疗脊髓损伤的实验研究

探讨自体骨髓干细胞( bone marrow stem cells, BMSC)动员移植与手术移植治疗脊髓损伤（spinal cord injury，SCI）的疗效和机制。方法：选用10周龄健康SD大鼠90只，雌雄各半，建模前注射5-溴2-脱氧尿嘧啶核苷 (Bromodeoxyuridine,Brdu) 50mg/kg/d×3天后抽取自体骨髓，体外分离自体BMSC；NYU(New York University，NYU) Impactor制作SCI模型，随机分为对照组、动员移植组、手术移植组各30只。动员移植组应用重组粒细胞刺激因子（granulocyte-colony stimulating factor,G-CSF）皮下注射，20mg/kg/d×7天；手术移植组为损伤局部移植0.3ml（1×107个/ml）BMSC,各组均从术前三天开始，连续10天腹腔注射Brdu 50mg/kg/d。采用Basso-Beattie-Bresnahan（BBB）评分检测大鼠后肢的运动功能；体感诱发电位（somatoseneory evoked potential,SEP）和运动诱发电位（motor evoke potential,MEP）检测脊髓上、下行神经传导通路，判断SCI和恢复程度；病理和免疫组化观察脊髓损伤组织细胞结构变化及Brdu、GFAP和NSE分布表达。结果：BBB评分1周以后动员移植组和手术移植组分别较对照组比较差异有统计学意义（p＜0.05）,SEP、MEP潜伏期和波幅2周后动员移植组和手术移植组较对照组比较差异有统计学意义，组织病理学显示动员移植组和手术移植组较对照组有更少的空洞、坏死及GFAP阳性胶质瘢痕组织，较多的Brdu阳性细胞和NSE阳性细胞。结论：自体BMSCs动员移植和手术移植两种方法均能明显减轻SCI的程度，促进损伤后的脊髓功能的恢复，二者对比，前者更为方便、无创，实用性强，更有可能抓住有限的治疗时机，因而应用前景可能更好。     

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。     

Combined transplantation of bone marrow mesenchymal stem cells and pedicled greater omentum promotes locomotor function and regeneration of axons after spinal cord injury in rats*★

BACKGROUND： According to previous studies, the neuroprotective effect of the pedicled greater omentum may be attributed to the secretion of neurotrophic factors and stimulation of angiogenesis. The neurotrophic factors released from the pedicled greater omentum, such as brain-derived neurotrophic factor and neurotrophin 3/4/5 could exert a neuroprotective effect on the damaged host neural and glial cells, and also could induce the transdifferentiation of transplanted bone marrow mesenchymal stem cells （BMSCs） into neural cells. OBJECTIVE： Based on the functions of the omentum of neuro-protection and vascularization, we hypothesize that the transplantation of BMSCs and pedicled greater omentum into injured rat spinal cord might improve the survival rate and neural differentiation of transplanted BMSCs and consequently gain a better functional outcome. DESIGN, TIME AND SETFING： A randomized, controlled animal experiment. The experiments were carried out at the Department of Anatomy, the Secondary Military Medical University of Chinese PLA between June 2005 and June 2007. MATERIALS： Fifteen male inbred Wistar rats, weighing （200±20） g, provided by the Experimental Animal Center of the Secondary Military Medical University of Chinese PLA were used and met the animal ethical standards. Mouse anti-BrdU and mouse anti-NF200 monoclonal antibody were purchased from Boster, China. METHODS： Cell culture： We used inbred Sprague-Dawley rats to harvest bone marrow for culture of BMSCs and transplantation to avoid possible immune rejection. BMSCs were cultured via total bone marrow adherence. Experimental grouping and intervention： The rats were randomly divided into a control group, cell group and combined group, five rats per group. Rats in the control group underwent spinal cord injury （SCI） only, during which an artery clamp with pressure force of 30 g was employed to compress the spinal cord at the Tl0 level for 30 seconds to produce the SCI model. 5 μ L PBS containing 10^5 BMSCs was injected in     

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.     

香芹酚对大鼠急性脊髓损伤的保护作用

目的 探讨香芹酚对大鼠脊髓损伤（SCI）后神经功能的影响及其机制。方法 将60只雄性SD大鼠（200~250 g）随机分为5组:假手术组（n=12）、SCI组（n=12）、香芹酚组（n=36）,香芹酚组根据香芹酚剂量分为低、中、高剂量3个亚组,每亚组12只。低、中、高剂量香芹酚组SCI后30 min腹腔注射香芹酚,剂量分别为10、20、40 mg/kg,每日一次;假手术组和SCI组每日腹腔注射等量生理盐水。采用Allen法建立大鼠SCI模型;假手术组只行椎板切除手术。SCI后24、48、72 h,采用BBB评分评估大鼠神经功能;SCI后72 h,采用ELISA法检测损伤脊髓组织丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽（GSH）、过氧化氢酶（CAT）、caspase-3活性;Western-blot法检测损伤脊髓组织Bax,Bcl-2蛋白表达水平。结果 SCI后,大鼠BBB评分均明显降低（P<0.05）,损伤脊髓组织水肿指数以及MDA、caspase-3和Bax水平均明显增高（P<0.05）,而SOD、GSH、CAT、Bcl-2水平均明显降低（P<0.05）;香芹酚能明显改善大鼠BBB评分（P<0.05）,明显降低水肿指数以及MDA、caspase-3和Bax水平（P<0.05）,而显著增加CAT、SOD、GSH、Bcl-2水平（P<0.05）。结论 香芹酚可通过减轻脊髓水肿、抑制氧化应激反应以及抗凋亡作用而对SCI大鼠发挥神经保护作用。     

Recovery of function following grafting of human bone marrow-derived stromal cells into the injured spinal cord

This study evaluates functional recovery after transplanting human bone marrow-derived stromal cells (BMSCs) into contusion models of spinal cord injury (SCI). The authors used a high-throughput process to expand BMSCs and characterized them by flow cytometry, ELISA, and gene expression. They found that BMSCs secrete neurotrophic factors and cytokines with therapeutic potential for cell survival and axon growth. In adult immune-suppressed rats, mild, moderate, or severe contusions were generated using the MASCIS impactor. One week following injury, 0.5 to 1 x 106 BMSCs were injected into the lesioned spinal cord; control animals received vehicle injection. Biweekly behavioral tests included the Basso, Beattie, and Bresnahan Locomotor Rating Scale (BBB), exploratory rearing, grid walking, and thermal sensitivity. Animals receiving moderate contusions followed by BMSC grafts showed significant behavioral recovery in BBB and rearing tests when compared to controls. Animals receiving BMSC grafts after mild or severe contusion showed trends toward improved recovery. Immunocytochemistry identified numerous axons passing through the injury in animals with BMSC grafts but few in controls. BMSCS were detected at 2 weeks after transplantation; however, at 11 weeks very few grafted cells remained. The authors conclude that BMSCs show potential for repairing SCI. However, the use of carefully characterized BMSCs improved transplantation protocols ensuring BMSC, survival, and systematic motor and sensory behavioral testing to identify robust recovery is imperative for further improvement.     

丹酚酸B促进大鼠急性脊髓损伤修复及量效关系探讨

目的 研究腹腔注射丹酚酸B(Sal B)对大鼠急性脊髓损伤(SCI)模型的神经保护作用和促功能恢复作用,探讨Sal B在急性SCI治疗的应用价值,并探讨其量效关系. 方法参照Allen法制作SD大鼠T9脊髓节段急性损伤模型,腹腔注射Sal B或PBS液,按照注射液的不同分为4组:Sal B高剂量组(20 mg/kg组),Sal B中剂量组(10 mg/kg组),Sal B低剂量组(2 mg/kg组)和对照组(注射PBS液),每组12只.用比色法检测髓过氧化物酶活性;用免疫组织化学染色法检测损伤脊髓节段MMP-1、c-Fos抗体表达情况,用干湿重法评价的水肿程度,并采用后肢功能评分(BBB)评分评价10 d内大鼠的运动功能恢复情况. 结果损伤后4 h Sal B治疗组髓过氧化物酶活性下降,损伤后1 dHE染色切片显示SalB组治疗后局部组织损伤减轻,炎性细胞浸润数量减少,损伤后1d免疫组化染色结果显示,Sal B治疗组比对照组MMP-1表达减少,c-Fos表达下调;Sal B治疗组水肿程度轻于对照组,从SCI后第7天起,SalB组高剂量组(20 mg/kg组)和对照组之间的BBB评分有显著性差异(P<0.05).各指标改善情况与Sal B剂量呈正相关性. 结论 Sal B可减轻大鼠SCI后的组织损伤,下调损伤相关因子MMP-1和c-Fos的表达,降低损伤局部髓过氧化物酶活性,减轻组织水肿,并能促进损伤大鼠的功能恢复.     

丹酚酸B促进大鼠急性脊髓损伤修复及量效关系探讨

目的 研究腹腔注射丹酚酸B(Sal B)对大鼠急性脊髓损伤(SCI)模型的神经保护作用和促功能恢复作用,探讨Sal B在急性SCI治疗的应用价值,并探讨其量效关系. 方法参照Allen法制作SD大鼠T9脊髓节段急性损伤模型,腹腔注射Sal B或PBS液,按照注射液的不同分为4组:Sal B高剂量组(20 mg/kg组),Sal B中剂量组(10 mg/kg组),Sal B低剂量组(2 mg/kg组)和对照组(注射PBS液),每组12只.用比色法检测髓过氧化物酶活性;用免疫组织化学染色法检测损伤脊髓节段MMP-1、c-Fos抗体表达情况,用干湿重法评价的水肿程度,并采用后肢功能评分(BBB)评分评价10 d内大鼠的运动功能恢复情况. 结果损伤后4 h Sal B治疗组髓过氧化物酶活性下降,损伤后1 dHE染色切片显示SalB组治疗后局部组织损伤减轻,炎性细胞浸润数量减少,损伤后1d免疫组化染色结果显示,Sal B治疗组比对照组MMP-1表达减少,c-Fos表达下调;Sal B治疗组水肿程度轻于对照组,从SCI后第7天起,SalB组高剂量组(20 mg/kg组)和对照组之间的BBB评分有显著性差异(P<0.05).各指标改善情况与Sal B剂量呈正相关性. 结论 Sal B可减轻大鼠SCI后的组织损伤,下调损伤相关因子MMP-1和c-Fos的表达,降低损伤局部髓过氧化物酶活性,减轻组织水肿,并能促进损伤大鼠的功能恢复.     

菲立磁标记兔BMSCs自体移植脊髓后的核磁共振活体示踪及形态学研究

目的通过观察菲立磁标记兔骨髓源性神经干细胞(BMSCs)自体移植脊髓后的核磁共振活体示踪及形态,期待找到一种应用非侵袭性方法来识别、跟踪BMSCs的存活状态及与宿主组织整合情况的方法。方法无菌条件下股骨取骨髓,梯度密度离心法分离获取兔骨髓基质细胞;使用“Feridex-多聚赖氨酸复合物(FE-PLL)”标记骨髓基质细胞,采用普鲁士兰染色和台盼蓝排除实验等方法鉴定FE-PLL标记兔骨髓基质细胞的效率和细胞的活力;体外标记的细胞自体脊髓移植,磁共振、免疫组织化学染色和透射电镜检查。结果普鲁士蓝染色显示FE-PLL标记骨髓基质细胞胞质内出现细小的蓝色铁颗粒;与正常未标记的细胞相比较,FE-PLL标记对骨髓基质细胞的活力、增殖和分化等能力没有明显的影响;经菲立磁标记的兔BMSCs自体脊髓移植后,可在核磁共振上活体示踪。结论菲立磁与核磁共振联合可无创性活体标记检测移植的神经干细胞基本的存在部位、存在方式及其一些生物学特性,可以用来活体示踪移植的BMSCs。     

Localization of bone marrow stromal cells in injured spinal cord treated by intravenous route depends on the hemorrhagic lesions in traumatized spinal tissues

OBJECTIVES: Bone marrow stromal cells (BMSCs) have been reported to improve movement deficit in adult rats with spinal cord injury (SCI). The purpose of this study is to determine the distribution of BMSCs in the spinal cord lesion of the contusion model of SCI. METHODS: Laminectomy was carried out at L1 vertebra level and SCI was carried out using the weight drop method. BMSCs were isolated from adult rats, labeled with bromodeoxyuridine (BrdU) and administered intravenously to the rats 1 week after SCI, which were killed after 4 weeks. The non-treated animals were used as negative control, which showed cavitations of the spinal cord after 5 weeks of SCI. Rats in another group were killed immediately and used to study the hemorrhagic lesions. The volume densities (Vv) of the hemorrhage and cavitation were the highest at the site of direct trauma. RESULTS: The numerical densities of the transmigrated cells per area (Nat) were as follows: 0.3 +/- 0.2, 3.9 +/- 0.4, 5.4 +/- 0.4, 8.4 +/- 0.5, 5.5 +/- 0.3, 3.6 +/- 0.3 and 0.4 +/- 0.2 at the end and the middle of the thoracic vertebra 13 (T13), the region between T13 and the first lumbar vertebra, the middle of L1, the region between L1 and L2, and the middle and the end of L2 vertebra, respectively. The distribution of Nat at the above regions was a Gaussian model. The volume densities of hemorrhage in the spinal cord taken from the above regions showed that hemorrhage with the highest volume density occurred at the impact site and the volume density declined as the samples taken were more distant from the impact site. DISCUSSION: The migration of BMSCs in the injured region depends on the amount of the hemorrhage and damage to blood vessels of the spinal cord.