不同方法共移植的嗅鞘细胞和雪旺氏细胞在损伤脊髓内的迁移和对轴突再生的影响

目的:观察经不同方法共移植的嗅鞘细胞和雪旺氏细胞在脊髓内的迁移特点和对轴突再生的影响.方法:将8只75±1d雌性SD大鼠随机分为2组,每组4只,利用NYU打击器制作TIO脊髓损伤模型,打击高度25mm,打击杆重量10g.造模后2周,一组采用联合移植,雪旺氏细胞移植于损伤鄢位中心,嗅鞘细胞移植于距离损伤中心0.5mm处的头侧和尾侧的脊髓中线上;另一组采用混合移植,将嗅鞘细胞和雪旺氏细胞混合后移植于距离损伤中心0.5mm处的头侧和尾侧的脊髓中线上.每个部位注射4个点,深度为1.75mm,1.25mm、1mm、0.5mm.注射速度为0.1μl/min.联合移植组嗅鞘细胞晕每点0.5μl含5×10~4个,雩旺氏细胞晕为每点1μl含10~5个雪旺氏细胞;混合移植组为每点1μl含嗅鞘细胞和雪旺氏细胞各5×10~4个.细胞移植后1周和8周时各组分别取2只大鼠.以损伤部位为中心取包含细胞移植部位的脊髓,荧光和共聚焦显微镜下观察细胞迁移情况,利用神经丝(neurofilment,NF)和乍长相关蛋白-43(growth associated protein-43,GAP-43)免疫荧光染色观察移植细胞对轴突再生的影响.结果:两组中均可见嗅鞘细胞迁移,主要在灰质和白质内沿脊髓纵轴向损伤部位迁移,还分别有一小部分沿中央管和蛛网膜下腔迁移;但雪旺氏细胞仅存与嗅鞘细胞混合移植于距离损伤中心0.5mm处的头侧和尾侧脊髓时才可见有限距离的迁移.联合移植时,NF阳性(NF+)和GAP-43阳性(GAP-43+)纤维伴随嗅鞘细胞迁移而沿脊髓纵轴延伸,雪旺氏细胞移植处可处NF+纤维从各个方向长入损伤部位(移植部位)并互相缠绕;混合移植时,大量NF+纤维伴随移植细胞迁移而延伸,损伤部佗虽然NF+纤维较少,但没有互相缠绕现象.结论:雪旺氏细胞在损伤脊髓内迁移能力差;嗅鞘细胞不仅具有良好的迁移能力,而且可促进雪旺氏细胞迁移.无论是联合移植还是混合移植,移植细胞均能促进轴突再生,但联合移植时雪旺氏细胞移植处再生纤维互相缠绕、无法延伸.     

经嗅鞘细胞条件培养液诱导的骨髓基质干细胞移植对脊髓损伤修复的影响

[目的]研究经嗅鞘细胞条件培养液诱导的骨髓基质干细胞(BMSCs)移植对脊髓损伤的治疗作用。[方法]采用脊髓挤压损伤方法,制造SD大鼠脊髓损伤动物模型(共24只),随机分成A、B、C三组(每组8只),立即分别将DMEM、BMSCs和经诱导的BMSCs移植入脊髓损伤部位,分别于移植4、8周后处死动物,均采用HE染色观察脊髓损伤处空洞面积改变情况;并采用抗神经丝(NF)抗体、生长相关蛋白-43(GAP-43)抗体和神经元特异性核蛋白(Neu N)抗体免疫组织化学荧光染色,观察移植的BMSCs存活和分化情况及损伤部位神经纤维再生情况。[结果]移植4、8周后,脊髓损伤区空洞为移植细胞充填,空洞面积明显减少,差异有统计学意义(P0.01),BBB评分结果 C组和A、B组之间存在统计学差异(P0.05)。B、C组均可见再生神经纤维形成,同时部分移植细胞呈Neu N染色阳性,在C组上述改变更加显著。[结论]经嗅鞘细胞条件培养液诱导的BMSCs可以在脊髓损伤区存活,能够显著减小脊髓损伤处空洞面积,并可以促进脊髓损伤区神经纤维的修复再生,促进大鼠双下肢运动功能的恢复。     

嗅鞘细胞移植调节星形细胞活性促进急性脊髓损伤大鼠神经功能恢复

[目的]探讨嗅鞘细胞(OECs)对急性脊髓损伤大鼠星形胶质细胞(AST)激活的影响。[方法]乳鼠嗅球制备嗅鞘细胞,制备急性脊髓损伤模型,随机分为3组,空白组:T_(9-11)椎板去除;对照组:T_(10)脊髓损伤,DMEM/F_(12)移植;实验组:T_(10)脊髓损伤,OEC移植。行BBB评分和体感诱发电位检测评价神经功能,Western Blot检测GFAP、CSPG、bFGF表达变化,观察脊髓损伤后星形胶质细胞激活情况及OEC移植对其影响。[结果](1)脊髓损伤后GFAP阳性细胞数目增加,实验组少于对照组,尼氏染色及NF-200阳性细胞数目实验组多于对照组,差异有统计学意义;(2)Western Blot:脊髓损伤后GFAP、CSPG表达增加,bFGF表达下降,OEC干预后,GFAP、CSPG表达较对照组下降。bFGF表达增加;(3)神经功能:脊髓损伤BBB评分及SEP显示治疗组大鼠神经功能得到改善。[结论]嗅鞘细胞移植可抑制星形胶质细胞增殖,调节因子分泌,促进大鼠脊髓损伤恢复。     

人胚胎嗅鞘细胞移植修复大鼠脊髓全横断损伤

[目的]探索人胚胎嗅鞘细胞(hOECs)移植修复大鼠脊髓全横断损伤的可行性。[方法]分离、培养并鉴定人胚嗅鞘细胞,24只W istar大鼠,随机分为实验组和对照组,均行T10节段脊髓全横断。术后第9~10 d,实验组、对照组分别移植Hoechst33342标记的hOECs 5μl(2.5×105个细胞)、DMEM-F12培养基5μl。移植术后第1、2、4、6、8、10周进行BBB运动功能评分,取材行荧光化学(Hoechst33342)和免疫组织化学染色(P75、NF-200、Syn-aptophysin)。双盲条件下进行数据统计。[结果]移植的hOECs可以在损伤脊髓内存活10周以上,可向损伤脊髓头尾两端迁移;术后4~10周实验组的BBB运动功能评分明显高于对照组(P<0.01);P75染色实验组呈阳性反应;NF-200和Synaptophysin免疫组化染色,实验组神经纤维、突触数目和密度都较对照组高。[结论]hOECs移植对脊髓全横断损伤大鼠运动功能恢复具有促进作用。     

嗅鞘细胞移植联合跑步训练对改善脊髓损伤大鼠后肢运动功能的研究

目的 :研究嗅鞘细胞移植联合跑步训练对改善脊髓损伤大鼠后肢运动功能的效果,同时探索嗅鞘细胞移植联合跑步训练改善大鼠运动功能的可能机制。方法:选用雄性SD大鼠80只,随机分为4组,每组20只,均进行脊髓损伤造模后进行如下分组处理:生理盐水注射组(A组)、嗅鞘细胞移植组(B组)、生理盐水注射联合跑步训练组(C组)和嗅鞘细胞移植联合跑步训练组(D组)。建模3 d后C、D两组进行跑步训练,1周后B、D两组进行嗅鞘细胞移植(每只大鼠注射总量为4μl,细胞浓度为1.0×106/μl),A、C两组给予相应剂量盐水。观察时间为4周,每组每周测量BBB评分,利用免疫组化染色观察Bax、Bcl-2及NF-200的表达,采用Mallory磷钨酸苏木素染色观察神经纤维数量,使用TUNEL染色观察神经元凋亡。结果:(1)BBB评分:嗅鞘细胞联合跑步训练组与其他3组在第4周差异有统计学意义(P0.05)。(2)在Bcl-2蛋白表达上,嗅鞘细胞移植联合跑步训练有交互作用,两者相互促进(P0.05);在Bax蛋白的表达上,嗅鞘细胞移植可以明显降低其表达,与跑步训练交互作用不明显(P0.05);TUNEL染色显示,嗅鞘细胞移植、跑步训练与时间因素三者具有交互作用,显著抑制细胞凋亡(P0.05)。(3)在Mallory磷钨酸苏木素染色及NF-200免疫组化染色上,嗅鞘细胞移植、跑步训练与时间因素三者具有交互作用(P0.05),促进神经再生。结论 :嗅鞘细胞移植联合跑步训练能够显著改善大鼠后肢运动功能,这可能通过以下途径实现:嗅鞘细胞移植与跑步训练能够相互协同,明显上调Bcl-2基因的表达,从而显著抑制神经元凋亡;同时能明显促进神经元轴索再生,提高神经纤维数量,并且这种效果可以随时间的延长更加显著。     

电针联合嗅鞘细胞移植对大鼠脊髓损伤神经轴突再生及走向的影响

目的:探讨电针对嗅鞘细胞(OECs)移植大鼠轴突再生的影响及作用机制。方法:2.5月龄Sprague Dawley(SD)雄性大鼠72只,体重(220±20)g,采用挫伤加全横断的造模方法造成T9脊髓损伤模型后随机分成模型组、电针组、嗅鞘细胞组和电针加嗅鞘细胞组。各组动物于造模后4周和8周注射5%荧光金水溶液(flurosecentgold,FG) 0.5 μl进行逆行标记,后进行荧光金逆行示踪观察以及动物行为学观察(BBB评分).结果:(1)BBB评分结果示术后第1天至第1周,各组间比较差异无统计学意义(P>0.05);从第3周开始,电针+OECs组高于模型组、OECs组、电针组3组(P<0.05).(2) 荧光金逆行示踪结果显示:术后4、8周各组脊髓内均可观察到有FG阳性神经纤维再生;在脊髓损伤区,电针加OECs组通过脊髓损伤区荧光金标记的阳性神经纤维数量多于其他3组,走行较其他3组规则。结论:电针联合OECs移植治疗能够极大的促进脊髓损伤大鼠神经纤维的再生以及大鼠后肢功能的恢复,能够恢复神经传导通路,并对再生的神经纤维生长方法有一定的导向性。     

静脉移植骨髓间充质干细胞对大鼠脊髓损伤后轴突再生的作用

目的:研究静脉移植骨髓间充质干细胞(bonemarrowstromalcells,BMSC)对脊髓损伤后轴突再生的影响。方法:体外分离、培养、扩增、纯化、BrdU标记10只同种异体SD大鼠的BMSC,WD法制成完全性截瘫的同种大鼠脊髓损伤动物模型。制模后7d随机分为A、B、C三组,A组22只,作为BMSC移植组,经鼠尾静脉移植2×106/ml的BMSC悬液1ml;B组22只,作为对照组,移植1mlL-DMEM液;C组22只,作为空白手术对照组。移植后1、2、3、4、5、6周用BBB评分评估各组后肢运动功能;移植后2、3、6周组织学及免疫组织化学观察并检测损伤段脊髓内生长相关蛋白-43(growth-associatedprotein-43,GAP-43)、神经丝蛋白200(neurofilament-200,NF200)的表达。结果:移植后3周开始,A组的BBB评分在各观察时间点明显高于B组、C组,移植后6周维持在较稳定水平,而B、C组在伤后3周开始维持在较稳定水平;在移植后2、3、6周,A组GAP-43、NF200的表达明显高于高于B组、C组,6周时开始下降,而B组与C组在移植后2、3、6周的表达均维持在相对恒定的水平。结论:大鼠脊髓损伤后静脉移植BMSC能使损伤段脊髓GAP-43、NF200表达上调,促进脊髓损伤后轴突再生和神经功能改善。     

人脐带间充质干细胞与自体激活雪旺细胞联合移植修复脊髓损伤的实验研究

目的 通过观察人脐带间充质干细胞(hUCMSCs)和大鼠自体激活雪旺细胞(AASCs)联合移植修复脊髓损伤的疗效,探讨AASCs对hUCMSCs体内存活、分化的影响.方法 分离、培养hUCMSCs和大鼠AASCs.通过IMPACTOR MODEL-Ⅱ型打击仪将80只Wistar成年雌性大鼠均制作成T10损伤模型,随机分为四组(n=20):DMEM移植对照组、hUCMSCs移植组、AASCs移植组、hUCMSCs与AASCs联合移植组.比较符组动物恢复情况,进行行为学评分(BBB评分),NF-200和GFAP染色观察细胞存活、分化情况,生物素葡聚糖胺示踪观察皮质脊髓束再生情况.结果 4周后各组间BBB评分差异有统计学意义(P<0.05),6周后联合移植组明显高于其他三组,差异有统计学意义(P<0.05).免疫组化染色示联合移植组的hUCMSCs存活数量,NF-200、GFAP阳性荧光面积均明显高于hUCMSCs移植组,差异有统计学意义(P<0.05),BDA顺行爪踪可见联合移植组于损伤区染色较多,部分纤维延续至损伤远端.结论 AASCs可支持移植的hUCMSCs在损伤部位存活并向神经方向分化,hUCMSCs与AASCs联合移植较二者单独移植能更有效地促进脊髓损伤后运动功能的恢复和轴突再生.     

软骨素酶联合雪旺细胞移植促进脊髓损伤修复的研究

目的 观察软骨素酶联合雪旺细胞移植在治疗急性脊髓损伤中的作用.方法 Wistar大鼠80只,制作T10节段急性脊髓损伤模型(致伤力10g×4cm).随机分成4组:对照组、雪旺细胞移植组、硫酸软骨素酶治疗组和联合治疗组.采用脊髓运动功能评分(BBB法,总分21)、神经电生理(SEP&MEP)检查和生物素葡聚糖胺(BDA)神经示踪及标本NF-200免疫组织化学染色等比较各组疗效.结果 4周后BBB评分实验组较对照组明显提高,各组间差异有统计学意义(P<0.05)(12周时4组分别为9.11±1.41、11.22±1.59、11.77±1.76和14.22±1.92).术后4周起,神经电生理检查实验组动物较对照组差异有统计学意义(P<0.05),12周时4组MEP的波幅分别恢复至术前的28.8%、44.9%、49.0%和56.8%.BDA示踪显示联合组较对照组有较多的神经纤维穿过损伤部位.NF-200免疫组织化学染色吸光度(A)比较各组间差异有统计学意义(P<0.05),3个实验组纵切片A值与对照组的比值为1.44、1.55和2.78.结论 联合应用软骨素酶和雪旺细胞移植来治疗脊髓损伤,起到协同作用,效果好于单一方法,能明显促进脊髓损伤后的轴突再生和肢体功能恢复.     

神经干细胞移植对大鼠脊髓损伤后BDNF与GAP-43基因表达的影响

目的:研究海马源性神经干细胞(NSCs)移植对大鼠脊髓损伤(SCI)后生长相关蛋白43(GAP-43)及脑源性神经营养因子(BDNF)基因表达的影响,探讨神经干细胞移植修复大鼠脊髓损伤的机制.方法:NSCs提取自新生胎鼠的海马区,经过培养及鉴定.实验分为3组:NSCs移植组、DMEM填充组、正常对照组.大鼠SCI后第7d移植NSCs,应用RTPCR法观察NSCs移植后,大鼠脊髓损伤区GAP-43和BDNF基因表达的变化.结果:NSCs移植组较单纯损伤组明显增强了GAP-43mRNA与BDNFmRNA的表达.结论:NSCs移植后改变脊髓损伤区的微环境,上调BDNFmRNA,促进GAP-43mRNA的表达,是修复脊髓损伤的机制之一.     

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.     

自体激活雪旺细胞联合胚胎脊髓细胞悬液修复脊髓损伤中的突触发育过程

 目的观察分析胚胎脊髓细胞悬液(fetal spinal cord cell suspension, FSCS)联合自体激活 雪旺细胞(autologus activated Schwann cells, AASCs)在损伤脊髓移植区中的突触发育过程。方法 42只 Wistar成年大鼠结扎单侧隐神经, 1周后取出结扎远端神经组织, 分离、培养、纯化 AASCs。以改良 Allen 法(10g x 5 cm)打击脊髓, 3天后将孕 14天(E₁₄) FSCS 20μl联合 AASCs植入损伤空腔, 移植后 2、4、6、 8、10和 12周, 以光学显微镜、电镜、免疫组织化学观察移植物成活、分化及其与宿主之间关系。结果 移植区 AASCs生长分化良好, 胶质瘢痕少。成神经细胞最先展示了胞质突起, 随之出现低电子密度的突 触前、后膜, 突触前、后膜电子密度逐渐增高形成良好的致密突起。突触小泡数量和种类逐渐增多, 突触 小泡有圆形清亮小泡、椭圆形小泡、颗粒状小泡和扁平小泡-f型。突触的连接方式由单个的胞体-树突 突触, 出现多个的胞体-树突和树突-树突突触。同时, 移植成神经细胞、成少突胶质细胞、成星形细胞的 细胞器日渐完善, 细胞功能活跃。血脑屏障也随之出现。移植区可见神经微丝、组织胺、降钙素基因相关 肽、胶原纤维酸性蛋白阳性纤维。结论 (1) AASCs辅助下 FSCS在成年大鼠损伤脊髓内可发育为成熟 的突触; (2) FSCS与宿主脊髓重建突触方式的信息交换具有潜在可行性。     

Axonal remyelination by cord blood stem cells after spinal cord injury

Human umbilical cord blood stem cells (hUCB) hold great promise for therapeutic repair after spinal cord injury (SCI). Here, we present our preliminary investigations on axonal remyelination of injured spinal cord by transplanted hUCB. Adult male rats were subjected to moderate SCI using NYU Impactor, and hUCB were grafted into the site of injury one week after SCI. Immunohistochemical data provides evidence of differentiation of hUCB into several neural phenotypes including neurons, oligodendrocytes and astrocytes. Ultrastructural analysis of axons reveals that hUCB form morphologically normal appearing myelin sheaths around axons in the injured areas of spinal cord. Colocalization studies prove that oligodendrocytes derived from hUCB secrete neurotrophic hormones neurotrophin-3 (NT3) and brain-derived neurotrophic factor (BDNF). Cord blood stem cells aid in the synthesis of myelin basic protein (MBP) and proteolipid protein (PLP) of myelin in the injured areas, thereby facilitating the process of remyelination. Elevated levels of mRNA expression were observed for NT3, BDNF, MBP and PLP in hUCB-treated rats as revealed by fluorescent in situ hybridization (FISH) analysis. Recovery of hind limb locomotor function was also significantly enhanced in the hUCB-treated rats based on Basso-Beattie-Bresnahan (BBB) scores assessed 14 days after transplantation. These findings demonstrate that hUCB, when transplanted into the spinal cord 7 days after weight-drop injury, survive for at least 2 weeks, differentiate into oligodendrocytes and neurons, and enable improved locomotor function. Therefore, hUCB facilitate functional recovery after moderate SCI and may prove to be a useful therapeutic strategy to repair the injured spinal cord.     

Neural stem cells and regeneration of injured spinal cord

Recent progress in the stem cell biology has led much insight into new therapeutic interventions aiming for the regeneration of the damaged central nervous system. The major strategies can be classified into two subgroups: (1) activation of endogenous neural stem cells, and (2) cell transplantation therapies. In either of these strategies, it is crucial to understand the underlying mechanisms of maintenance, activation, and differentiation of neural stem cells and subsequent process, including the migration, survival, and functional maturation of differentiated cells. In this paper, we would like to summarize our recent findings on the therapeutic interventions of the injured spinal cord, especially focusing on the development of treatment for the acute phase of spinal cord injury with anti-interleukin (IL)-6 receptor blocking antibody.     

Autologous mesenchymal stem cells in chronic spinal cord injury

Spinal cord injury (SCI) occurs in the most productive part of life. Treatment options for treatment of chronic SCI are few and have limited impact on clinical outcome. Central nervous system (CNS) has limited intrinsic regeneration capability. The study included patients with chronic complete SCI. Previously harvested autologous mesenchymal stem cells were administered at the site of injury after a laminectomy. Follow-up was done by a neutral examiner not involved in the surgery every 3 months. One patient had improvement in motor power. Two patients had a patchy improvement in pin prick sensation below the level of injury. Three different, progressively increasing doses did not result in improvement in the clinical outcome. Though the administration of allogenic human mesenchymal stem cells is safe in patients with SCI, it may not be efficacious; especially in patients with chronic SCI.     

人脐血单个核细胞促进脊髓损伤后的轴突再生

[目的] 讨论人脐血单个核细胞(human cord blood mononuclear cells,HCMNCs)移植治疗大鼠脊髓损伤后损伤区域轴突再生情况和功能恢复.[方法] 利用 Impactor Model Ⅱ打击器制成30例T10脊髓损伤模型,分组为:实验对照组(DMEM细胞培养基),损伤后3 d HCMNCs移植组,损伤后14 d HCMNCs移植组,每组10例.以HE染色和免疫组化染色以及BDA顺行示踪染色观察脊髓损伤处轴突再生情况,结合对各组实验动物脊髓损伤后肢体功能的恢复情况进行行为学评分(BBB 评分),综合评估脊髓功能恢复程度.[结果] 与对照组比较,HCMNCs移植治疗能够明显促进神经轴突再生,改善功能恢复,损伤后14 d HCMNCs移植组优于损伤后3 d移植组,各组间疗效差异具有统计学意义(P<0.01).[结论] HCMNCs在体内向神经元及神经胶质细胞分化,促进神经轴突再生和功能恢复.损伤后14 d是移植的较为理想的时间.     

Effect of human neural progenitor cells on injured spinal cord

Human central nervous system ( CNS) has verylimited regenerative potentials. Patients withsevere injuries in the CNS such as spinal cordinjury (SCI) frequently endure lifelong disability. Avariety of methods have been tried to prevent spinalcord from further injury and foster regeneration afterSCI. Despite these efforts, an effective treatment forthis disease is still lacking. Since neural progenitorcells have already committed to become neural cells inthe CNS, they appear to be a good c…     

Trauma-induced tumorigenesis of cells implanted into the rat spinal cord

OBJECT: Findings in several clinical cases have suggested a correlation between tumor formation and previous injury to the central nervous system (CNS); however, the relationship between trauma and tumorigenesis has not been investigated well experimentally. In this study the authors provide evidence correlating tumorigenesis with trauma in the rat spinal cord. METHODS: A glial cell line, C6R-G/H, which expresses green fluorescent protein (GFP) and hygromycin phosphotransferase (HPT), was implanted into normal and injured rat spinal cords. In all rats in which the cells were implanted into an injured site, locomotor function deteriorated and histological analysis demonstrated glioblastoma multiforme by 6 weeks; tumorigenesis was correlated with a loss of both GFP expression and resistance to hygromycin treatment. In contrast, no evidence of tumor formation was found at 6 weeks in rats in which the cells were implanted into healthy tissue. When C6R-G/H cells were treated with contused spinal cord extract in culture before implantation, they lost GFP expression and hygromycin resistance, and later formed tumors after implantation into normal spinal cord. CONCLUSIONS: The findings of this study indicate that trauma can induce tumorigenesis. Implantation of C6R-G/H cells into traumatized spinal cords resulted in their transformation, which was signaled by loss of GFP expression and hygromycin resistance accompanied by tumor formation. Exposure to extracts derived from injured spinal cord produced similar transformation and gene expression changes, as well as tumor formation after such cells were implanted into normal cords. Care, therefore, should be taken when cells are implanted into an injured CNS because of potential mutagenesis due to trauma-induced factors.